Nicotinamide promotes cardiomyocyte derivation and survival through kinase inhibition in human pluripotent stem cells.

Nicotinamide, the amide form of Vitamin B3, is a common nutrient supplement that plays important role in human fetal development. Nicotinamide has been widely used in clinical treatments, including the treatment of diseases during pregnancy. However, its impacts during embryogenesis have not been fully understood. In this study, we show that nicotinamide plays multiplex roles in mesoderm differentiation of human embryonic stem cells (hESCs). Nicotinamide promotes cardiomyocyte fate from mesoderm progenitor cells, and suppresses the emergence of other cell types. Independent of its functions in PARP and Sirtuin pathways, nicotinamide modulates differentiation through kinase inhibition. A KINOMEscan assay identifies 14 novel nicotinamide targets among 468 kinase candidates. We demonstrate that nicotinamide promotes cardiomyocyte differentiation through p38 MAP kinase inhibition. Furthermore, we show that nicotinamide enhances cardiomyocyte survival as a Rho-associated protein kinase (ROCK) inhibitor. This study reveals nicotinamide as a pleiotropic molecule that promotes the derivation and survival of cardiomyocytes, and it could become a useful tool for cardiomyocyte production for regenerative medicine. It also provides a theoretical foundation for physicians when nicotinamide is considered for treatments for pregnant women.

Bufalin-induced cardiotoxicity: new findings into mechanisms.

Bufalin is one of the main pharmacological and toxicological components of Venenum Bufonis and many traditional Chinese medicine preparations. The cardiotoxicity clearly limits its application to patients living in countries. Hence, an investigation of its toxicological mechanism is helpful for new drug development and treatment of the related clinical adverse reactions. We investigate the cardiotoxicity of bufalin using human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) (0.003-0.1 µmol·L-1), human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) (0.03-0.3 µmol·L-1) and eight human cardiac ion channel currents (0.01-100 µmol·L-1) combined with an impedance-based bioanalytical and patch clamp method. Biphasic effect of bufalin on the contractility in hiPSC-CMs, which has been shown to strengthen myocardial contractility, accelerate conduction, and increase beating rate at the earlier stage of administration, whereas weakened myocardial contractility, abolished conduction, and ceased beating rate at the later stage of administration. Bufalin decreased the action potential duration (Action potential duration at 30%, 50% and 90% repolarization), cardiac action potential amplitude, and maximal depolarization rate and depolarized the resting membrane potential of hiPSC-CMs. Spontaneous beating rates of hiPSC-CMs were markedly increased at 0.03 µmol·L-1, while were weakened at 0.3 µmol·L-1 after application. Bufalin blocks INav1.5 in a concentration-dependent manner with half maximal inhibitory concentration of 74.5 µmol·L-1. Bufalin respectively increased the late sodium current and Na+-Ca2+ exchange current with a concentration for 50% of maximal effect of 2.48 and 66.06 µmol·L-1 in hiPSC-CMs. Whereas, bufalin showed no significant effects on other cardiac ion channel currents. The enhancement of the late sodium current is one of the main mechanism for cardiotoxicity of bufalin.

Rapid Electrical Stimulation Increased Cardiac Apoptosis Through Disturbance of Calcium Homeostasis and Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

BACKGROUND/AIMS: Heart failure induced by tachycardia, the most common arrhythmia, is frequently observed in clinical practice. This study was designed to investigate the underlying mechanisms. METHODS: Rapid electrical stimulation (RES) at a frequency of 3 Hz was applied on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for 7 days, with 8 h/day and 24 h/day set to represent short-term and long-term tachycardia, respectively. Age-matched hiPSC-CMs without electrical stimulation or with slow electrical stimulation (1 Hz) were set as no electrical stimulation (NES) control or low-frequency electrical stimulation (LES) control. Following stimulation, JC-1 staining flow cytometry analysis was performed to examine mitochondrial conditions. Apoptosis in hiPSC-CMs was evaluated using Hoechst staining and Annexin V/propidium iodide (AV/PI) staining flow cytometry analysis. Calcium transients and L-type calcium currents were recorded to evaluate calcium homeostasis. Western blotting and qPCR were performed to evaluate the protein and mRNA expression levels of apoptosis-related genes and calcium homeostasis-regulated genes. RESULTS: Compared to the controls, hiPSC-CMs following RES presented mitochondrial dysfunction and an increased apoptotic percentage. Amplitudes of calcium transients and L-type calcium currents were significantly decreased in hiPSC-CMs with RES. Molecular analysis demonstrated upregulated expression of Caspase3 and increased Bax/Bcl-2 ratio. Genes related to calcium re-sequence were downregulated, while phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) was significantly upregulated following RES. There was no significant difference between the NES control and LES control groups in these aspects. Inhibition of CaMKII with 1 µM KN93 partly reversed these adverse effects of RES. CONCLUSION: RES on hiPSC-CMs disturbed calcium homeostasis, which led to mitochondrial stress, promoted cell apoptosis and caused electrophysiological remodeling in a time-dependent manner. CaMKII played a central role in the damages induced by RES, pharmacological inhibition of CaMKII activity partly reversed the adverse effects of RES on both structural and electrophysiological properties of cells.

Idiopathic isolated fibrotic atrial cardiomyopathy underlies unexplained scar-related atrial tachycardia in younger patients.

Aims: Unexplained scar-related atrial tachycardia (AT) has been frequently encountered in clinical practice. We hypothesized that idiopathic, isolated fibrotic atrial cardiomyopathy (ACM) underlies this rhythm disorder. This study was aimed to characterize the underlying substrate and to explore the aetiology of this unexplained scar-related AT. Methods and results: Twenty-six (11 men, aged 46 ± 13 years) of 52 non-surgical scar-related AT patients identified by three-dimensional voltage mapping were enrolled in this prospective observational study. Multimodality image examinations (echocardiography, cardiac magnetic resonance, 99Tc single-photon emission computed tomography), ventricular voltage mapping, and intracardiac pressure curve recording ruled out ventricular involvement. Catheter ablation was acutely successful for all the patients, and pacemaker implantation was performed in seven patients who presented sinus node dysfunction or atrial standstill after termination of the AT. In three patients with multiple AT recurrences, the diseased areas of the right atrium were resected and dechannelled via mini-invasive surgical interventions. Histological examinations revealed profound fibrosis without amyloidosis or adipose deposition. Viral and familial investigations yielded negative results. Fibrosis progression over a median of 45 (5-109) months of follow-up manifested as atrial arrhythmia recurrence in seven patients and atrial lead non-capture due to newly developed atrial standstill in two patients. Two patients suffered four ischaemic stroke events before receiving anticoagulation treatment. Conclusion: Isolated, fibrotic ACM may underlie the idiopathic scar-related ATs. This novel cardiomyopathy has unique clinical characteristics with high morbidity including stroke and warrants specific therapeutic strategies. Further investigations are required to determine the aetiology and mechanism.

The chemokine CXCL9 exacerbates chemotherapy-induced acute intestinal damage through inhibition of mucosal restitution.

PURPOSE: Acute intestinal damage induced by chemotherapeutic agent is often a dose-limiting factor in clinical cancer therapy. The aim of this study was to investigate the effect of chemokine CXCL9 on the intestinal damage after chemotherapy and explore the therapeutic potential of anti-CXCL9 agents. METHODS: In vitro cell proliferation assay was performed with a non-tumorigenic human epithelial cell line MCF10A. Multiple pathway analysis was carried out to explore the pathway that mediated the effect of CXCL9, and the corresponding downstream effector was identified with enzyme-linked immunosorbent assays. Chemotherapy-induced mouse model of intestinal mucositis was prepared by a single injection of the chemotherapeutic agent 5-fluorouracil (5-FU). In vivo expression of cxcl9 and its receptor cxcr3 in intestinal mucosa after chemotherapy was determined by quantitative real-time PCR. Therapeutic treatment with anti-CXCL9 antibodies was investigated to confirm the hypothesis that CXCL9 can contribute to the intestinal epithelium damage induced by chemotherapy. RESULTS: CXCL9 inhibited the proliferation of MCF10A cells by activating phosphorylation of p70 ribosomal S6 kinase (p70S6K), which further promotes the secretion of transforming growth factor beta (TGF-ß) as the downstream effector. A blockade of phospho-p70S6K with inhibitor abolished the effect of CXCL9 on MCF10A cells and reduced the secretion of TGF-ß. The expression levels of cxcl9 and cxcr3 were significantly up-regulated in intestinal mucosa after 5-FU injection. Neutralizing elevated CXCL9 with anti-CXCR9 antibodies successfully enhanced reconstitution of intestinal mucosa and improved the survival rate of mice that received high-dose chemotherapy. CONCLUSIONS: CXCL9 inhibits the proliferation of epithelial cells via phosphorylation of p70S6K, resulting in the excretion of TGF-ß as downstream mediator. CXCL9/CXCR3 interaction can exacerbate chemotherapeutic agent-induced intestinal damage, and anti-CXCL9 agents are potential novel therapeutic candidates for promoting mucosal restitution.