Journal: Life

Article Title: Identification of miRNAs as Biomarkers of Cardiac Protection in Non-Genetically Modified Primary Human Cardiomyocytes Exposed to Halogenated Hypnotics in an In Vitro Model of Transfection and Ischemia/Reperfusion: A New Model in Translational Anesthesia

doi: 10.3390/life13010064

Figure Lengend Snippet: Primary human cardiomyocytes 24 h post-transfection: ( A ) non-transfected HCMs, 10× optical microscopy objective; ( B ) non-transfected HCMs, 10× fluorescence microscopy image; ( C ) transfected HCMs, 10× optical microscopy objective; ( D ) transfected HCMs, 10× fluorescence microscopy objective. HCMs were transfected with 3 µL of TransIT-siQUEST ® transfection reagent and miRNA Mimic Negative Control-FAM, to a final concentration of 5 nM.

Article Snippet: This protocol was developed with primary human cardiomyocytes isolated from normal human ventricular tissue of the adult heart acquired from PromoCell ® ; however, it could be adapted to primary human cardiomyocytes isolated from fresh tissue prepared according to the standard procedures described in several published studies [ ].

Techniques: Transfection, Microscopy, Fluorescence, Negative Control, Concentration Assay

Journal: Regenerative Therapy

Article Title: Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells

doi: 10.1016/j.reth.2015.08.001

Figure Lengend Snippet: Detection of undifferentiated hiPSCs in primary cardiomyocytes using the LIN28 /qRT-PCR method. (A) LIN28 , NANOG , REX1 , SOX2 , OCT3/4 , KLF4 and c-MYC relative mRNA expression in cardiomyocytes was determined using qRT-PCR analysis. (B) qRT-PCR analysis of hiPSCs spiked into three lots of primary cardiomyocytes. Single-cell hiPSCs (0.1%, 1 × 10 3 cells; 0.01%, 1 × 10 2 cells; 0.001%, 1 × 10 1 cells) were spiked into 1 × 10 6 primary cardiomyocytes, and total RNA was isolated from the mixed cells. All values are expressed as mRNA levels relative to those in undifferentiated hiPSCs. hCM: human cardiomyocyte. The expression levels of target genes were normalized to those of the GAPDH transcript. Results are presented as the mean ± standard deviation (n = 3).

Article Snippet: In the spike experiments, 253G1 cells and primary human cardiomyocytes (PromoCell) were mixed at a defined cell number before RNA isolation. qRT-PCR was performed with the QuantiTect Probe one-step RT-PCR Kit (Qiagen) on a 7300 Real-Time PCR System (Applied Biosystems).

Techniques: Quantitative RT-PCR, Expressing, Isolation, Standard Deviation

Journal: Regenerative Therapy

Article Title: Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells

doi: 10.1016/j.reth.2015.08.001

Figure Lengend Snippet: Detection of undifferentiated hiPSCs in hiPSC-derived cardiomyocytes using the LIN28 /qRT-PCR method. (A) Schematic diagram of culture procedure for cardiomyocyte. (B) qRT-PCR analysis of cardiomyocyte markers, TNNT2 , GATA4 , NKX2.5 and MYH6 . Total RNA was isolated from hiPSC-derived cardiomyocytes (black bar) and primary cardiomyocytes (white bar). The mRNA levels are shown relative to those in primary cardiomyocytes. (C) Flow cytometry analysis of TNNT2 in hiPSCs (red), and hiPSC-derived cardiomyocytes at day 20 (blue). (D) LIN28 expression in hiPSCs differentiating into cardiomyocytes (d10 and d20). LIN28 mRNA levels are shown relative to that in undifferentiated hiPSCs. hCM: human cardiomyocyte. The expression levels of target genes were normalized to those of the GAPDH transcript. Results are presented as the mean ± standard deviation (n = 3).

Article Snippet: In the spike experiments, 253G1 cells and primary human cardiomyocytes (PromoCell) were mixed at a defined cell number before RNA isolation. qRT-PCR was performed with the QuantiTect Probe one-step RT-PCR Kit (Qiagen) on a 7300 Real-Time PCR System (Applied Biosystems).

Techniques: Derivative Assay, Quantitative RT-PCR, Isolation, Flow Cytometry, Expressing, Standard Deviation

Journal: Regenerative Therapy

Article Title: Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells

doi: 10.1016/j.reth.2015.08.001

Figure Lengend Snippet: Detection of undifferentiated hiPSCs in primary cardiomyocytes using the LIN28 /ddPCR method. (A) The copy number of LIN28 mRNA in 0.5 and 0.05 ng total RNA of hiPSC was investigated by ddPCR. (B) The copy number of LIN28 mRNA in 50 ng total RNA of hiPSC-spiked cardiomyocytes and three lots of primary cardiomyocytes were investigated by ddPCR. Dots indicate the fluorescence intensity of the droplets. Droplets were assigned “positive” or “negative” based on their fluorescence amplitude. (C) Raw droplet data shown in (B) were quantified as LIN28 mRNA copies. These samples used in Fig. 1 B and Fig. 3B/C were same ones. hCM: human cardiomyocyte. Results are presented as the mean ± standard deviation (n = 3).

Article Snippet: In the spike experiments, 253G1 cells and primary human cardiomyocytes (PromoCell) were mixed at a defined cell number before RNA isolation. qRT-PCR was performed with the QuantiTect Probe one-step RT-PCR Kit (Qiagen) on a 7300 Real-Time PCR System (Applied Biosystems).

Techniques: Fluorescence, Standard Deviation

Journal: European Heart Journal

Article Title: Effects of canagliflozin on human myocardial redox signalling: clinical implications

doi: 10.1093/eurheartj/ehab420

Figure Lengend Snippet: Direct effects of canagliflozin on human cardiomyocytes (hCM). Canagliflozin (10 μΜ) induced phosphorylation of AMPK and acetyl-coA carboxylase (ACC) in high glucose (HG)-treated human cardiomyocytes ( A and B, n = 8) and increased BH4 levels without affecting total biopterin levels ( C–E, n = 8). Canagliflozin decreased basal, NADPH-stimulated and Vas2870-inhibitable O 2 . − and increased the value of L-NAME delta( O 2 . − ) in human cardiomyocytes ( F–I, n = 7). Dihydroethidium (DHE) staining combined with Vas2870 confirmed these ( J–L, n = 8). Data are presented as mean ± SD. P -values are calculated by Wilcoxon signed-rank test ( A–H, J, K ) and paired t -test ( I ).

Article Snippet: The effects of SGLT1/2 on cardiomyocytes were first screened in the rat cardiac myocyte-derived cell line, H9c2, and the key findings were replicated in human cardiomyocytes (hCM, PromoCell, Heidelberg, Germany).

Techniques: Staining

Journal: European Heart Journal

Article Title: Effects of canagliflozin on human myocardial redox signalling: clinical implications

doi: 10.1093/eurheartj/ehab420

Figure Lengend Snippet: SGLT1 mediates the effects of canagliflozin on myocardial redox state. Canagliflozin increased ADP/ATP dose-dependently in human cardiomyocytes, while glucose deprivation from the culture medium (NG) induced similar changes in ADP/ATP ratio ( A, n = 8). There were no differences in AMPK and ACC phosphorylation among the NG-incubated cells with or without canagliflozin (10μΜ), and high glucose (HG)-incubated cells with canagliflozin (panels B and C, n = 8). SGLT1 expression was detected, while SGLT2 was not detected in human cardiomyocytes ( D–F, n = 8). SGLT1 was knocked down using siRNA (transfection low toxicity and efficiency was confirmed by transfecting BlockiT Alexa Fluor Red Fluorescent control, G ), resulting into ∼76% down-regulation of SGLT1 mRNA (panel H, n = 6), and ∼85% protein down-regulation (panel I, n = 6). Canagliflozin-induced AMPK and ACC phosphorylation was attenuated in SGLT1 knocked down human cardiomyocytes compared with siRNA ctrl human cardiomyocytes ( J and K, n = 8). SGLT1 deletion diminished canagliflozin-induced decrease of basal ( L ), NADPH-stimulated ( M ), and Vas2870-inhibitable O 2 . − production ( N ) and increased L-NAME delta O 2 . − ( O ). n = 8 in L–O . Data are presented as mean ± SD. P -values are calculated by Wilcoxon signed-rank test ( A–C, H, I ). Comparisons in canagliflozin responses between siControl and siSGLT1 cells were performed with two-way ANOVA with treatment (canagliflozin) × cell type (siControl or siSGLT1) interaction ( J–O ).

Article Snippet: The effects of SGLT1/2 on cardiomyocytes were first screened in the rat cardiac myocyte-derived cell line, H9c2, and the key findings were replicated in human cardiomyocytes (hCM, PromoCell, Heidelberg, Germany).

Techniques: Incubation, Expressing, Transfection

Journal: European Heart Journal

Article Title: Effects of canagliflozin on human myocardial redox signalling: clinical implications

doi: 10.1093/eurheartj/ehab420

Figure Lengend Snippet: Canagliflozin has a global anti-inflammatory and anti-apoptotic effect on human cardiomyocytes. In this experiment, human cardiomyocytes were cultured in high-glucose medium (25 mM) for 72 h and then treated with canagliflozin (10 μM) or DMSO for 24 h. Heat map of 127 down- or up-regulated genes by canagliflozin (fold change >1.5 or `−1.5, P < 0.05) in canagliflozin-treated human cardiomyocytes from n = 7 patients ( A ). NFkB, Wnt, IL1, IL3, TNFα, chemokine, MAPK pathways, and apoptotic pathways (highlighted by red font) were down-regulated by canagliflozin (more than 50% of pathway genes down-regulated by canagliflozin, B ). TNFRSF11, TRAF5, FZD7, CASP7 , and BAD were the most down-regulated individual genes upon canagliflozin treatment, implicated in NFkB, TNFα, and apoptosis pathways. The mRNA expression of these genes was positively correlated with myocardial NADPH oxidase activity (i.e. high Vas2870-inhibitable signal) in 240 atrial biopsies from Study 1 ( C ). Data are presented as median [25th–75th percentile]. P -values are calculated by Mann–Whitney U -test for high (above median) vs. low (below median).

Article Snippet: The effects of SGLT1/2 on cardiomyocytes were first screened in the rat cardiac myocyte-derived cell line, H9c2, and the key findings were replicated in human cardiomyocytes (hCM, PromoCell, Heidelberg, Germany).

Techniques: Cell Culture, Expressing, Activity Assay, MANN-WHITNEY

Journal: Biomedicines

Article Title: Saxagliptin Cardiotoxicity in Chronic Heart Failure: The Role of DPP4 in the Regulation of Neuropeptide Tone

doi: 10.3390/biomedicines10071573

Figure Lengend Snippet: Effect of DPP4 inhibition and/or neuropeptide substrates on the viability of AC16 cells. Western blot analysis of DPP4 ( A ) in healthy human left ventricle samples and AC16 cells. In vitro treatment protocol with various gliptins on AC16 cell line and cell viability (calcein assay) results ( B ). In vitro treatment protocol with neuropeptides and their combined administration with saxagliptin and their effect on the viability of AC16 cells ( C ). One-way ANOVA, Tukey’s post hoc test, and unpaired t -test. Data are presented as mean ± SEM. Group sizes: ( B ) n = 3 from 1 independent experiment, ( C ) n = 7 from 7 independent experiments. RFU: relative fluorescence unit.

Article Snippet: Human cardiac myocyte AC16 cell line was obtained from Merck (SCC109).

Techniques: Inhibition, Western Blot, In Vitro, Fluorescence