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 2.5µM). 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.

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.

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.

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.