Phosphoproteomics reveals a novel mechanism underlying the proarrhythmic effects of nilotinib, vandetanib, and mobocertinib.

The use of tyrosine kinase inhibitors (TKIs) has resulted in significant occurrence of arrhythmias. However, the precise mechanism of the proarrhythmic effect is not fully understood. In this study, we found that nilotinib (NIL), vandetanib (VAN), and mobocertinib (MOB) induced the development of "cellrhythmia" (arrhythmia-like events) in a concentration-dependent manner in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Continuous administration of NIL, VAN, or MOB in animals significantly prolonged the action potential durations (APD) and increased susceptibility to arrhythmias. Using phosphoproteomic analysis, we identified proteins with altered phosphorylation levels after treatment with 3 µM NIL, VAN, and MOB for 1.5 h. Using these identified proteins as substrates, we performed kinase-substrate enrichment analysis to identify the kinases driving the changes in phosphorylation levels of these proteins. MAPK and WNK were both inhibited by NIL, VAN, and MOB. A selective inhibitor of WNK1, WNK-IN-11, induced concentration- and time-dependent cellrhythmias and prolonged field potential duration (FPD) in hiPSC-CMs in vitro; furthermore, administration in guinea pigs confirmed that WNK-IN-11 prolonged ventricular repolarization and increased susceptibility to arrhythmias. Fingding indicated that WNK1 inhibition had an in vivo and in vitro arrhythmogenic phenotype similar to TKIs. Additionally,three of TKIs reduced hERG and KCNQ1 expression at protein level, not at transcription level. Similarly, the knockdown of WNK1 decreased hERG and KCNQ1 protein expression in hiPSC-CMs. Collectively, our data suggest that the proarrhythmic effects of NIL, VAN, and MOB occur through a kinase inhibition mechanism. NIL, VAN, and MOB inhibit WNK1 kinase, leading to a decrease in hERG and KCNQ1 protein expression, thereby prolonging action potential repolarization and consequently cause arrhythmias.

Transcriptomics coupled with proteomics reveals osimertinib-induced myocardial mitochondrial dysfunction.

Osimertinib, an irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) used for cancer treatment, can cause significant cardiac toxicity. However, the specific mechanism of osimertinib-induced cardiotoxicity is not fully understood. In this study, we administered osimertinib to mice and neonatal rat ventricular myocytes (NRVMs). We observed significant structural and functional damage to the hearts of these mice, along with a marked increase in cardiac injury biomarkers and accompanying ultrastructural damage to mitochondria. We integrated 4D label-free protein quantification and RNA-Seq methods to analyze the sequencing data of NRVMs under osimertinib treatment (0 and 2.5 µM). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis evidenced that differentially expressed genes (DEGs)and differentially expressed proteins (DEPs) were distinctly enriched for oxidative phosphorylation (OXPHOs). Simultaneously, osimertinib primarily affected the contents of adenosine triphosphate (ATP). Further investigations revealed that osimertinib disrupts the functions of the ATP synthase (complex V), leading to a reduction in ATP production. Taken together, our data demonstrated that osimertinib causes mitochondrial dysfunction, which in turn leads to the onset of cardiac toxicity.

Berberine attenuates sunitinib-induced cardiac dysfunction by normalizing calcium regulation disorder via SGK1 activation.

Sunitinib (SNT)-induced cardiotoxicity is associated with abnormal calcium regulation caused by phosphoinositide 3 kinase inhibition in the heart. Berberine (BBR) is a natural compound that exhibits cardioprotective effects and regulates calcium homeostasis. We hypothesized that BBR ameliorates SNT-induced cardiotoxicity by normalizing the calcium regulation disorder via serum and glucocorticoid-regulated kinase 1 (SGK1) activation. Mice, neonatal rat cardiomyocytes (NRVMs), and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to study the effects of BBR-mediated SGK1 activity on the calcium regulation disorder caused by SNT as well as the underlying mechanism. BBR offered prevention against SNT-induced cardiac systolic dysfunction, QT interval prolongation, and histopathological changes in mice. After the oral administration of SNT, the Ca2+ transient and contraction of cardiomyocytes was significantly inhibited, whereas BBR exhibited an antagonistic effect. In NRVMs, BBR was significantly preventive against the SNT-induced reduction of calcium transient amplitude, prolongation of calcium transient recovery, and decrease in SERCA2a protein expression; however, SGK1 inhibitors resisted the preventive effects of BBR. In hiPSC-CMs, BBR pretreatment significantly prevented SNT from inhibiting the contraction, whereas coincubation with SGK1 inhibitors antagonized the effects of BBR. These findings indicate that BBR attenuates SNT-induced cardiac dysfunction by normalizing the calcium regulation disorder via SGK1 activation.

Pharmacological suppression of Nedd4-2 rescues the reduction of Kv11.1 channels in pathological cardiac hypertrophy.

The human ether-á-go-go-related gene (hERG) encodes the pore-forming subunit (Kv11.1), conducting a rapidly delayed rectifier K+ current (I Kr). Reduction of I Kr in pathological cardiac hypertrophy (pCH) contributes to increased susceptibility to arrhythmias. However, practical approaches to prevent I Kr deficiency are lacking. Our study investigated the involvement of ubiquitin ligase Nedd4-2-dependent ubiquitination in I Kr reduction and sought an intervening approach in pCH. Angiotensin II (Ang II) induced a pCH phenotype in guinea pig, accompanied by increased incidences of sudden death and higher susceptibility to arrhythmias. Patch-clamp recordings revealed a significant I Kr reduction in pCH cardiomyocytes. Kv11.1 protein expression was decreased whereas its mRNA level did not change. In addition, Nedd4-2 protein expression was increased in pCH, accompanied by an enhanced Nedd4-2 and Kv11.1 binding detected by immunoprecipitation analysis. Cardiac-specific overexpression of inactive form of Nedd4-2 shortened the prolonged QT interval, reversed I Kr reduction, and decreased susceptibility to arrhythmias. A synthesized peptide containing the PY motif in Kv11.1 C-terminus binding to Nedd4-2 and a cell-penetrating sequence antagonized Nedd4-2-dependent degradation of the channel and increased the surface abundance and function of hERG channel in HEK cells. In addition, in vivo administration of the PY peptide shortened QT interval and action potential duration, and enhanced I Kr in pCH. We conclude that Nedd4-2-dependent ubiquitination is critically involved in I Kr deficiency in pCH. Pharmacological suppression of Nedd4-2 represents a novel approach for antiarrhythmic therapy in pCH.

Downregulation of hERG channel expression by tyrosine kinase inhibitors nilotinib and vandetanib predominantly contributes to arrhythmogenesis.

Cardiotoxicity by tyrosine kinase inhibitors remains an important concern. Nilotinib and vandetanib clinically carry high proarrhythmic risk and the exact mechanism underlying arrhythmogenesis is not fully understood. In this study, we investigated the effects of nilotinib and vandetanib on the abundance of human ether-á-go-go-related gene (hERG) K+ channel and assessed the potential role of acute hERG blockage versus chronic effects in arrhythmogenesis. We found that both nilotinib and vandetanib prolonged the field potential duration reflecting the repolarisation process and induced cellrythmias of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in a time-and concentration-dependent manner after, after chronic exposure. Patch-clamp recordings revealed significant reductions of hERG current densities by nilotinib or vandetanib after chronic incubation with hERG-HEK293 cells in addition to the acute inhibition. Western blot analysis showed that nilotinib and vandetanib decreased mature hERG protein (155-kDa) expression, in a greater extent than that of the immature form (135-kDa). A serum and glucocorticoid kinase 1 (SGK1) activator, C4-ceramide, prevented the nilotinib-and vandetanib-induced hERG protein downregulation and thus the incidence of cellrrhythmias. Taken together, our data demonstrated that the downregulation of hERG channel abundance on the cellular membrane predominantly contributed to the proarrhythmic effect of nilotinib and vandetanib.

Inhibitory G-protein-mediated modulation of slow delayed rectifier potassium channels contributes to increased susceptibility to arrhythmogenesis in aging heart.

BACKGROUND: Slow delayed rectifier potassium current (IKs) is an important component of repolarization reserve during sympathetic nerve excitement. However, little is known about age-related functional changes of IKs and its involvement in age-dependent arrhythmogenesis. OBJECTIVE: The purpose of this study was to investigate age-related alteration of the IKs response to ß-adrenergic receptor (ßAR) activation. METHODS: Dunkin-Hartley guinea pigs were used. Whole-cell patch-clamp recording was used to record K+ currents. Optical mapping of membrane potential was performed in ex vivo heart. RESULTS: There was no difference in IKs density in ventricular cardiomyocytes between young and old guinea pigs. However, in contrast to IKs potentiation in young hearts, isoproterenol (ISO) evoked an acute inhibition on IKs in a concentration-dependent manner in old guinea pig hearts. The ß2AR antagonist, but not ß1AR antagonist, reversed the inhibitory response. Preincubation of cardiomyocytes with the inhibitory G protein (Gi) inhibitor pertussis toxin (PTX) also reversed the inhibitory response. In HEK293 cells cotransfected with cloned IKs channel and ß2AR, ISO enhanced the current but reduced it when cells were cotransfected with Gi2, and PTX restored the ISO-induced excitatory response. Moreover, in aging cardiomyocytes, Gßγ inhibitor gallein, PLC inhibitor U73122, or protein kinase C inhibitor Bis-1 prevented the reduction of IKs by ISO. Furthermore, cardiac-specific Gi2 overexpression in young guinea pigs predisposed the heart to ventricular tachyarrhythmias. PTX pretreatment protected the hearts from ventricular arrhythmias. CONCLUSION: ßAR activation acutely induces an inhibitory IKs response in aging guinea pig hearts through ß2AR-Gi signaling, which contributes to increased susceptibility to arrhythmogenesis in aging hearts.

Downregulation of miR-146a Contributes to Cardiac Dysfunction Induced by the Tyrosine Kinase Inhibitor Sunitinib.

The main adverse effect of tyrosine kinase inhibitors, such as sunitinib, is cardiac contractile dysfunction; however, the molecular mechanisms of this effect remain largely obscure. MicroRNAs (miRNAs) are key regulatory factors in both cardiovascular diseases and the tyrosine kinase pathway. Therefore, we analyzed the differential expression of miRNAs in the myocardium in mice after exposure to sunitinib using miRNA microarray. A significant downregulation of miR-146a was observed in the myocardium of sunitinib-treated mice, along with a 20% decrease in left ventricle ejection fraction (LVEF). The downregulation of miR-146a was further validated by RT-qPCR. Among the potential targets of miR-146a, we focused on Pln and Ank2, which are closely related to cardiac contractile dysfunction. Results of luciferase reporter assay confirmed that miR-146a directly targeted the 3' untranslated region of Pln and Ank2. Significant upregulation of PLN and ANK2 at the mRNA and protein levels was observed in the myocardium of sunitinib-treated mice. Cardiac-specific overexpression of miR-146a prevented the deteriorate effect of SNT on calcium transients, thereby alleviating the decreased contractility of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). SiRNA knockdown of PLN or ANK2 prevented sunitinib-induced suppression of contractility in hiPSC-CMs. Therefore, our in vivo and in vitro results showed that sunitinib downregulated miR-146a, which contributes to cardiac contractile dysfunction by regulating the downstream targets PLN and ANK2, and that upregulation of miR-146a alleviated the inhibitory effect of SNT on cardiac contractility. Thus, miR-146a could be a useful protective agent against sunitinib-induced cardiac dysfunction.

Upregulation of phosphoinositide 3-kinase prevents sunitinib-induced cardiotoxicity in vitro and in vivo.

Sunitinib (SNT) is a multi-targeted receptor tyrosine kinase inhibitor that has been approved by the FDA for cancer therapy. However, its cardiotoxicity has limited the clinical applicability with no effective therapeutic approach available. As a broadband kinase inhibitor, the function of several kinases that are essential to cardiac function might also be affected by SNT, such as calmodulin-dependent protein kinase (CaMKII), cyclic-AMP-dependent protein kinases (PKA), AMP-activated protein kinase (AMPK), and phosphoinositide 3 kinase (PI3K). In this study, we investigated whether SNT-induced cardiotoxicity could be prevented by blocking SNT-induced alteration in the corresponding signaling pathways. In human induced pluripotent stem cell-derived cardiomyocytes, SNT (0.5-20 µmol/L) inhibited contractility of cardiomyocytes in a concentration-dependent manner, and the inhibitory effect was prevented either by PIP3 (1 µmol/L) application or PI3K overexpression. On the contrary, the CaMKII inhibitor KN-93 (50 nmol/L), PKA inhibitor H89 (1 µmol/L), and AMPK activators metformin (2 mmol/L) and 5-aminoimidazole-4-carboxamide 1-b-D-ribofuranoside (2 mmol/L) presented negligible effects. Oral SNT administration (40 mg/kg/day) in mice progressively decreased the PI3K activity and cardiac function in 2 weeks with a significant decrease in the expression and activity of Cav1.2 and SERCA. Cardiac-specific PI3K overexpression through adeno-associated virus 9-mediated gene delivery in mice prevented SNT-induced reduction in cardiac function, calcium transient, calcium current, and Cav1.2 expression. In summary, our data indicate that increased PI3K activity is protective against SNT-induced calcium mishandling and contractile dysfunction. Cardiac-specific PI3K activation could be an effective therapeutic approach to treat SNT cardiotoxicity in patients with cancer.

The role of Na(+), K(+)-ATPase in the hypoxic vasoconstriction in isolated rat basilar artery.

Hypoxia-induced cerebrovascular dysfunction is a key factor in the occurrence and the development of cerebral ischemia. Na(+), K(+)-ATPase affects the regulation of intracellular Ca(2+) concentration and plays an important role in vascular smooth muscle function. However, the potential role of Na(+), K(+)-ATPase in hypoxia-induced cerebrovascular dysfunction is unknown. In this study, we found that the KCl-induced contraction under hypoxia in rat endothelium-intact basilar arteries is similar to that of denuded arteries, suggesting that hypoxia may cause smooth muscle cell (SMC)-dependent vasoconstriction in the basilar artery. The Na(+), K(+)-ATPase activity of the isolated basilar artery with or without endothelium significantly reduced with prolonged hypoxia. Blocking the Na(+)-Ca(2+) exchanger with Ni(2+) (10(-3)M) or the L-type Ca(2+) channel with nimodipine (10(-8)M) dramatically attenuated KCl-induced contraction under hypoxia. Furthermore, prolonged hypoxia significantly reduced Na(+), K(+)-ATPase activity and increased [Ca(2+)]i in cultured rat basilar artery SMCs. Hypoxia reduced the protein and mRNA expression of the α2 isoform of Na(+), K(+)-ATPase in SMCs in vitro. We used a low concentration of the Na(+), K(+)-ATPase inhibitor ouabain, which possesses a high affinity for the α2 isoform. The contractile response in the rat basilar artery under hypoxia was partly inhibited by ouabain pretreatment. The decreased Na(+), K(+)-ATPase activity in isolated basilar artery and the increased [Ca(2+)]i in SMCs induced by hypoxia were partly inhibited by pretreatment with a low concentration of ouabain. These results suggest that hypoxia may educe Na(+), K(+)-ATPase activity in SMCs through the α2 isoform contributing to vasoconstriction in the rat basilar artery.

Hydrogen sulfide suppresses outward rectifier potassium currents in human pluripotent stem cell-derived cardiomyocytes.

AIM: Hydrogen sulfide (H2S) is a promising cardioprotective agent and a potential modulator of cardiac ion currents. Yet its cardiac effects on humans are poorly understood due to lack of functional cardiomyocytes. This study investigates electrophysiological responses of human pluripotent stem cells (hPSCs) derived cardiomyocytes towards H2S. METHODS AND RESULTS: Cardiomyocytes of ventricular, atrial and nodal subtypes differentiated from H9 embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were electrophysiologically characterized. The effect of NaHS, a donor of H2S, on action potential (AP), outward rectifier potassium currents (I(Ks) and I(Kr)), L-type Ca²âº currents (I(CaL)) and hyperpolarization-activated inward current (I(f)) were determined by patch-clamp electrophysiology and confocal calcium imaging. In a concentration-dependent manner, NaHS (100 to 300 µM) consistently altered the action potential properties including prolonging action potential duration (APD) and slowing down contracting rates of ventricular-and atrial-like cardiomyocytes derived from both hESCs and hiPSCs. Moreover, inhibitions of slow and rapid I(K) (I(Ks) and I(Kr)), I(CaL) and I(f) were found in NaHS treated cardiomyocytes and it could collectively contribute to the remodeling of AP properties. CONCLUSIONS: This is the first demonstration of effects of H2S on cardiac electrophysiology of human ventricular-like, atrial-like and nodal-like cardiomyocytes. It reaffirmed the inhibitory effect of H2S on I(CaL) and revealed additional novel inhibitory effects on I(f), I(Ks) and I(Kr) currents in human cardiomyocytes.

One-step derivation of cardiomyocytes and mesenchymal stem cells from human pluripotent stem cells.

Cardiomyocytes (CMs) and mesenchymal stem cells (MSCs) are important cell types for cardiac repair post myocardial infarction. Here we proved that both CMs and MSCs can be simultaneously generated from human induced pluripotent stem cells (hiPSCs) via a pro-mesoderm differentiation strategy. Two hiPSC lines, hiPSC (1) and hiPSC (2) were generated from human dermal fibroblasts using OCT-4, SOX-2, KLF-4, c-Myc via retroviral-based reprogramming. H9 human embryonic stem cells (hESCs) served as control. CMs and MSCs were co-generated from hiPSCs and hESCs via embryoid body-dependent cardiac differentiation protocol involving a serum-free and insulin-depleted medium containing a p38 MAPK inhibitor, SB 203580. Comparing to bone marrow and umbilical cord blood-derived MSCs, hiPSC-derived MSCs (iMSCs) expressed common MSC markers and were capable of adipogenesis, osteogenesis and chondrogenesis. Moreover, iMSCs continuously proliferated for more than 32 population doublings without cellular senescence and showed superior pro-angiogenic and wound healing properties. In summary, we generated a large number of homogenous MSCs in conjunction with CMs in a low-cost and efficient one step manner. Functionally competent CMs and MSCs co-generated from hiPSCs may be useful for autologous cardiac repair.

Scutellarin blocks sodium current in freshly isolated mouse hippocampal CA1 neurons.

Scutellarin (Scu), the main bioactive component of Erilgeron breviscapus, protects the brain against ischemic damages. To explore the therapeutic mechanism of Scu, we investigated the impact of Scu on sodium current (I(Na)) of freshly isolated mouse hippocampal CA1 neurons using the whole-cell patch clamp technique. Results showed that Scu inhibited I(Na) in concentration- and holding potential-dependent manners. At 50 µM, Scu markedly shifted the steady state inactivation curve of I (Na) towards a more negative potential, slowed down the recovery of I(Na) from inactivation state, and elicited a frequency-dependent block of I (Na). The shape of the current-voltage (I-V) curve and the steady state activation curve of I(Na) were unaffected by Scu treatment. These findings suggest that Scu is capable of inhibiting I(Na) in neurons through predominantly affecting the inactivated state of I(Na). Inhibition of Na(+) channels provides a novel pharmacological basis for the anti-ischemic application of Scu.