Background The prevalence of heart failure is constantly increasing, and the prognosis of patients remains poor. New treatment strategies to preserve cardiac function and limit cardiac hypertrophy are therefore urgently needed. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used as an experimental platform for cardiac in vitro studies. However, in contrast to adult cardiomyocytes, hiPSC-CMs display immature morphology, contractility, gene expression and metabolism and hence express a naive phenotype that resembles more of a foetal cardiomyocyte. Methods A library of 14 novel compounds was synthesized in-house and screened for GATA4-NKX2-5 reporter activity and cellular toxicity. The most potent compound, 3i-1262, along with previously reported GATA4-acting compounds, were selected to investigate their effects on hypertrophy induced by endothelin-1 or mechanical stretch. Morphological changes and protein expression were characterized using immunofluorescence staining and high-content analysis. Changes in gene expression were studied using qPCR and RNA sequencing. Results The prototype compound 3i-1262 inhibited GATA4-NKX2-5 synergy in a luciferase reporter assay. Additionally, the isoxazole compound 3i-1262 inhibited the hypertrophy biomarker B-type natriuretic peptide (BNP) by reducing BNP promoter activity and proBNP expression in neonatal rat ventricular myocytes and hiPSC-CMs, respectively. Treatment with 3i-1262 increased metabolic activity and cardiac troponin T expression in hiPSC-CMs without affecting GATA4 protein levels. RNA sequencing analysis revealed that 3i-1262 induces gene expression related to metabolic activity and cell cycle exit, indicating a change in the identity and maturity status of hiPSC-CMs. The biological processes that were enriched in upregulated genes in response to 3i-1262 were downregulated in response to mechanical stretch, and conversely, the downregulated processes in response to 3i-1262 were upregulated in response to mechanical stretch. Conclusions There is currently a lack of systematic understanding of the molecular modulation and control of hiPSC-CM maturation. In this study, we demonstrated that the GATA4-interfering compound 3i-1262 reorganizes the cardiac transcription factor network and converts hypertrophic signalling towards enhanced cardiomyocyte identity and maturity. This conceptually unique approach provides a novel structural scaffold for further development as a modality to promote cardiomyocyte specification and maturity.

Abstract Introduction Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. Patient-derived human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an informative platform to study disease pathomechanisms. Transcription factor GATA4 serves a significant role in cardiomyocyte hypertrophy, and GATA4-targeted compounds inhibit hypertrophy [1]. Purpose This study aims at identifying phenotypic differences between patient-derived hiPSC-CMs and control hiPSC-CMs and at investigating the effect of a novel GATA4-targeting compound C-2021 on the patient-derived hiPSC-CMs phenotype. Methods hiPSC-CMs were differentiated from a healthy, control line (iPS(IMR90)-4; here, control hiPSC-CMs) and from HCM patient-derived hiPSCs (HCM hiPSC-CMs) that carry a mutation in the MYBPC3 gene. Two types of hypertrophic stimuli were applied to hiPSC-CMs: mechanical stretch and endothelin-1 (ET-1) treatment. For pharmacological assays, compound C-2021 (at 30 µM) or vehicle (0.1 % DMSO) were introduced to hiPSC-CMs one hour prior to the stimuli. hiPSC-CMs were exposed to cyclic equibiaxial mechanical stretching for 24 h or 72 h using Flexcell FX-5000 Tension System (Flexcell International Corporation) [2]. The expression of hypertrophic markers (such as NPPB, NPPA) and structural proteins (including α-myosin and β-myosin heavy chains; MYH6 and MYH7, respectively) was investigated with qPCR. The relative gene expression was analysed using the 2-ΔΔCt method using 18S and ACTB as reference genes. Furthermore, hiPSC-CMs were subjected to ET-1 at 100 nM for 24 h and proBNP expression was quantified using high-content analysis (HCA). Results HCM hiPSC-CMs displayed increased basal expression of NPPB (Fig. 1A-B), lower expression of MYH6 (Fig. 1C) and higher expression of MYH7 (Fig. 1D) compared to control hiPSC-CMs, which is in line with the hypertrophic disease phenotype. Control and HCM hiPSC-CMs responded to 24 h and 72 h mechanical stretching by increased NPPB expression (Fig. 1A-B). At 72 h, the increase in NPPB expression was more pronounced in the HCM hiPSC-CMs (Fig. 1B). The compound C-2021 inhibited stretch-induced NPPB expression after 72 h stretching. HCA results indicate that ET-1 induced a 17-fold increase in proBNP-positive control hiPSC-CMs, and this effect was attenuated by C-2021 treatment. Interestingly, ET-1 did not enhance proBNP expression in HCM hiPSC-CMs (Fig. 2). Conclusions Our results show that the HCM hiPSC-CMs differ from the control hiPSC-CMs in response to hypertrophic stimuli and in myosin heavy chain isoform expression. Additionally, C-2021 may have anti-hypertrophic effects in both in control and HCM hiPSC-CMs based on the downregulation of NPPB expression, and the decrease in proBNP-positive hiPSC-CMs. Further studies are needed to elucidate the exact mechanisms underlying HCM and the action of C-2021 on the HCM hiPSC-CM phenotype.

We have no conflict of interest to disclose concerning this work.

Pre-analytical factors are an important source of variation or errors in clinical laboratory measurements. Based on the new accreditation standards, medical and laboratory professions now seek to develop tools to deal systematically with these diverse factors. Several obvious pre-analytical uncertainty components were estimated in pragmatic experiments and combined with data on analytical variation and literature knowledge on biological variation, to estimate the measurement uncertainty of most common chemical and haematological examinations in clinical laboratories. The main aim was to assess quality specifications for regional laboratory services. The expanded measurement uncertainties (level of confidence 95%) of serum cholesterol, albumin and potassium remained within 13-16%. The major uncertainty component for cholesterol was biological variation, whereas those for albumin and potassium were sample collection and pretreatment. The measurement uncertainties for serum free thyroxin, thyrotropin and C-reactive protein, 20%, 42% and 125% respectively, were largely due to their biological variation. The measurement uncertainties of basic erythrocyte parameters (erythrocyte count and mean corpuscular volume, blood haemoglobin concentration) were less than 10%. Larger measurement uncertainties were obtained for thrombocyte and leukocyte counts, 24 and 31%, respectively, and for the reticulocyte fraction, 41%.

BACKGROUND:Preservatives that could prevent destruction of cells, casts, and bacteria in urine are of great practical importance because they allow centralization and improvement of accuracy of urine particle counting. We compared two in-house mixtures and one commercial solution, as well as refrigeration, for their ability to preserve urine for both automated analysis (flow cytometry) and visual microscopy.METHODS:Urine specimens were preserved by refrigeration at 4 degrees C without preservatives (procedure 1); in a lyophilized solution intended to preserve specimens for bacterial culture (Urine C&S tubes; BD Preanalytical Solutions; procedure 2); in 10 mL/L formalin-0.15 mol/L NaCl (procedure 3); in 80 mL/L ethanol-20 g/L polyethylene glycol (procedure 4); and by storage at 20 degrees C without preservatives (procedure 5). Test strip measurements were used to select specimens positive for leukocyte esterase, hemoglobin, albumin, or nitrite. For 106 consecutive strip-positive specimens, urinalysis was performed by UF-100 (Sysmex) and by phase-contrast microscopy after Sternheimer supravital staining. Automated analysis was performed at arrival in the morning, on the same day in the afternoon, and after 1 and 3 days. Visual microscopy was performed at arrival and 3 days later.RESULTS:Urine bacterial counts were well preserved with procedures 1-3, with a false-positive rate of 0.0-3.4% at day 3 vs 28% without preservation (procedure 5). Erythrocytes were poorly preserved for 3 days (kappa coefficients, 0.24-0.61); after 1 day, fair preservation was seen with procedure 2 (kappa = 0.78), compared with less favorable preservation with procedure 1 (kappa = 0.61) or procedure 5 (kappa = 0.66). Leukocytes were well preserved by all five procedures in the acidic adult urines investigated. Counts of casts and large epithelial cells were artifactually increased by procedure 3. Procedure 2 performed at least as well as refrigeration for specimens analyzed with visual microscopy.CONCLUSIONS:Urine specimens from adults can be stabilized at room temperature for both automated particle analysis and visual microscopy.