Supplementary MaterialsSupplemental Material1 – Supplemental materials for The cytoprotective effect of yes-associated protein 1 after ischemia-reperfusion injury in AC16 human cardiomyocytes Supplemental_Material1. – Supplemental material for The cytoprotective impact of yes-associated protein 1 after ischemia-reperfusion injury in AC16 human cardiomyocytes Supplemental_Material4.pdf (4.7K) GUID:?29B86DDB-E499-4B0D-8677-2FD06963B837 Supplemental material, Supplemental Material4 for The cytoprotective impact of yes-associated protein 1 after ischemia-reperfusion injury in AC16 human cardiomyocytes by Kashif Khan, Georges Makhoul, Bin Yu, Adel Schwertani and Renzo Cecere in Experimental Biology and Medicine Supplemental Material5 – Supplemental material for The cytoprotective impact of yes-associated protein 1 after ischemia-reperfusion injury in AC16 human cardiomyocytes Supplemental_Material5.pdf (11K) GUID:?DE3244B0-D2AA-4D8D-B5FB-A7053ADFD5D8 Supplemental material, Supplemental Material5 for The cytoprotective impact of yes-associated protein 1 after ischemia-reperfusion injury in AC16 human cardiomyocytes by Kashif Khan, Georges Makhoul, Bin Yu, Adel Schwertani and Renzo Cecere in Experimental Biology and Medicine Short CCF642 abstract The Hippo-signaling pathway is a mechanism implicated in cardiomyocyte cytoprotection and regeneration after a myocardial infarction. Yes-associated protein 1, the main effector protein of this pathway, acts as a co-transcriptional activator to promote cardiomyocyte proliferation and survival. However, the biological mechanisms by which yes-associated protein 1 protects the heart post-MI are currently unknown. Here, we propose that yes-associated protein 1 plays a critical role in cardiomyocyte CCF642 cytoprotection after simulated ischemia-reperfusion injury. AC16 human cardiomyocytes were infected with lentiviral plasmids containing normal human yes-associated protein 1 and a constitutively active form of YAP, YAP1S127A. Cells were exposed to ischemia-reperfusion injury using a hypoxic chamber. Hippo-signaling characterization after ischemia-reperfusion injury was performed via Western blotting and reverse transcriptase polymerase chain reaction. Cell viability, apoptosis, and cellular hypertrophy were assessed as a measure of cytoprotection. The GSK3 inhibitor CHIR99021 was used to investigate cross-talk between Hippo and Wnt-signaling and their role in cytoprotection after ischemia-reperfusion-injury. Ischemia-reperfusion injury resulted in significant decreased expression of the non-phosphorylated Hippo signaling kinases MST1 and LATS1, along with decreased expression of YAP/TAZ. Overexpression of yes-associated protein 1 improved cellular viability, while reducing hypertrophy and apoptosis via the ATM/ATR DNA damage response pathway. Activation of -catenin in YAP-infected cardiomyocytes synergistically reduced cellular hypertrophy after ischemia-reperfusion-injury. Our findings indicate that yes-associated protein 1 is cytoprotective in AC16 human cardiomyocytes after ischemia-reperfusion injury, which might be mediated by co-activation from the canonical Wnt/-catenin pathway. Therefore, activation of yes-associated proteins 1 could be a book restorative to correct the infarcted myocardium. Effect statement Genetically executive the cells from the center after myocardial infarction to show a far more regenerative phenotype can be a guaranteeing therapy for center failure patients. Right here, we support a regenerative part for yes-associated proteins 1, the primary effector proteins from the Hippo signaling pathway, in AC16 human being cardiomyocytes like a potential restorative gene focus on for cardiac restoration after myocardial infarction. style of ischemia-reperfusion (IR) damage. Using Traditional western blotting and change transcriptase polymerase string reaction (RT-PCR), we investigated the noticeable modification in expression of Hippo-signaling proteins after IR injury. We used a lentiviral-based strategy to overexpress YAP1 in AC16 human being cardiomyocytes and evaluated its results on cell viability, apoptosis, oxidative tension, and hypertrophy. Finally, to research the cross-talk between Hippo and Wnt signaling, we activated both -catenin and YAP1 in AC16 human cardiomyocytes and assessed their effects on cellular hypertrophy after IR injury. Materials and methods Cell culture CCF642 The AC16 human left ventricular cardiomyocytes (Millipore Sigma) were used for this study. AC16 cells stained positively for several cardiac markers including atrial natriuretic peptide, brain natriuretic peptide, a-catenin, myosin heavy chain 7, troponin I, connexin 43, and GATA4.29 Cells were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient F-12 Ham (DMEM/F-12) (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin. Cells at passages three to five were used throughout the study. Generation of YAP1-lentiviral vectors Two lentiviral vectors promoting the overexpression of YAP1 in AC16 human cardiomyocytes were cloned into entry vectors made up of a green fluorescence protein (GFP) gene, under the mouse phosphoglycerate kinase 1 promoter, and a puromycin resistance gene, under the CMV promoter. Gateway recombination was used to generate a flag-tagged YAP1 plasmid (YAP) and a flag-tagged YAP-mutant that is resistant FLJ34463 to phosphorylation at the 127 serine position (YAPS127A), thus promoting its nuclear entry.30 Plasmid sequences were confirmed via polymerase chain reaction (PCR) (Supplemental Desk 1, Supplemental Body CCF642 1(a)) and genomic sequencing, that was performed on the McGill Gnome and University Qubec Innovation Center, Montral, Canada (data not proven). 293T capable cells had been gifted by Dr. Trembley (McGill College or university, Montreal, Canada) and had been.