Translation reinitiation in c.453delC frameshift mutation of KCNH2 producing functional hERG K+ channels with mild dominant negative effect in the heterozygote patient-derived iPSC cardiomyocytes.

The c.453delC (p.Thr152Profs*14) frameshift mutation in KCNH2 is associated with an elevated risk of Long QT syndrome (LQTS) and fatal arrhythmia. Nevertheless, the loss-of-function mechanism underlying this mutation remains unexplored and necessitates an understanding of electrophysiology. To gain insight into the mechanism of the LQT phenotype, we conducted whole-cell patch-clamp and immunoblot assays, utilizing both a heterologous expression system and patient-derived induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) with 453delC-KCNH2. We also explored the site of translational reinitiation by employing LC/MS mass spectrometry. Contrary to the previous assumption of early termination of translation, the findings of this study indicate that the 453delC-KCNH2 leads to an N-terminally truncated hERG channel, a potential from a non-canonical start codon, with diminished expression and reduced current (IhERG). The co-expression with wildtype KCNH2 produced heteromeric hERG channel with mild dominant-negative effect. Additionally, the heterozygote patient-derived iPSC-CMs exhibited prolonged action potential duration and reduced IhERG, which was ameliorated with the use of a hERG activator, PD-118057. The results of our study offer novel insights into the mechanisms involved in congenital LQTS associated with the 453delC mutation of KCNH2. The mutant results in the formation of less functional N-terminal-truncated channels with reduced amount of membrane expression. A hERG activator is capable of correcting abnormalities in both the heterologous expression system and patient-derived iPSC-CMs.

Impact of High-Dose Irradiation on Human iPSC-Derived Cardiomyocytes Using Multi-Electrode Arrays: Implications for the Antiarrhythmic Effects of Cardiac Radioablation.

Cardiac radioablation is emerging as an alternative option for refractory ventricular arrhythmias. However, the immediate acute effect of high-dose irradiation on human cardiomyocytes remains poorly known. We measured the electrical activities of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) upon irradiation with 0, 20, 25, 30, 40, and 50 Gy using a multi-electrode array, and cardiomyocyte function gene levels were evaluated. iPSC-CMs showed to recover their electrophysiological activities (total active electrode, spike amplitude and slope, and corrected field potential duration) within 3-6 h from the acute effects of high-dose irradiation. The beat rate immediately increased until 3 h after irradiation, but it steadily decreased afterward. Conduction velocity slowed in cells irradiated with ≥25 Gy until 6-12 h and recovered within 24 h; notably, 20 and 25 Gy-treated groups showed subsequent continuous increase. At day 7 post-irradiation, except for cTnT, cardiomyocyte function gene levels increased with increasing irradiation dose, but uniquely peaked at 25-30 Gy. Altogether, high-dose irradiation immediately and reversibly modifies the electrical conduction of cardiomyocytes. Thus, compensatory mechanisms at the cellular level may be activated after the high-dose irradiation acute effects, thereby, contributing to the immediate antiarrhythmic outcome of cardiac radioablation for refractory ventricular arrhythmias.

The antioxidant icariin protects porcine oocytes from age-related damage in vitro.

OBJECTIVE: If fertilization does not occur within a specific period, the quality of unfertilized oocytes in the oviduct (in vivo aging) or in culture (in vitro aging) will deteriorate over time. Icariin (ICA), found in all species of Epimedium herbs, has strong antioxidant activity, and is thought to exert anti-aging effects in vitro. We asked whether ICA protects oocytes against age-related changes in vitro. METHODS: We analyzed the reactive oxygen species (ROS) levels and expression of antioxidant, maternal, and estrogen receptor genes, and along with spindle morphology, and the developmental competence and quality of embryos in the presence and absence of ICA. RESULTS: Treatment with 5 µM ICA (ICA-5) led to a significant reduction in ROS activity, but increased mRNA expression of glutathione and antioxidant genes (superoxide dismutase 1 [SOD1], SOD2, peroxiredoxin 5, and nuclear factor erythroid 2-like 2), during aging in vitro. In addition, ICA-5 prevented defects in spindle formation and chromosomal alignment, and increased mRNA expression of cytoplasmic maturation factor genes (bone morphogenetic protein 15, cyclin B1, MOS proto-oncogene, serine/threonine kinase, and growth differentiation factor-9). It also prevented apoptosis, increased mRNA expression of antiapoptotic genes (BCL2-like 1 and baculoviral IAP repeat-containing 5), and reduced mRNA expression of pro-apoptotic genes (BCL2 antagonist/killer 1 and activation of caspase-3). Although the maturation and cleavage rates were similar in all groups, the total cell number per blastocyst and the percentage of apoptotic cells at the blastocyst stage were higher and lower, respectively, in the control and ICA-5 groups than in the aging group. CONCLUSION: ICA protects oocytes against damage during aging in vitro; therefore, it can be used to improve assisted reproductive technologies.

Antiviral activity and safety of remdesivir against SARS-CoV-2 infection in human pluripotent stem cell-derived cardiomyocytes.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is considered as the most significant global public health crisis of the century. Several drug candidates have been suggested as potential therapeutic options for COVID-19, including remdesivir, currently the only authorized drug for use under an Emergency Use Authorization. However, there is only limited information regarding the safety profiles of the proposed drugs, in particular drug-induced cardiotoxicity. Here, we evaluated the antiviral activity and cardiotoxicity of remdesivir using cardiomyocytes-derived from human pluripotent stem cells (hPSC-CMs) as an alternative source of human primary cardiomyocytes (CMs). In this study, remdesivir exhibited up to 60-fold higher antiviral activity in hPSC-CMs compared to Vero E6 cells; however, it also induced moderate cardiotoxicity in these cells. To gain further insight into the drug-induced arrhythmogenic risk, we assessed QT interval prolongation and automaticity of remdesivir-treated hPSC-CMs using a multielectrode array (MEA). As a result, the data indicated a potential risk of QT prolongation when remdesivir is used at concentrations higher than the estimated peak plasma concentration. Therefore, we conclude that close monitoring of the electrocardiographic/QT interval should be advised in SARS-CoV-2-infected patients under remdesivir medication, in particular individuals with pre-existing heart conditions.

Antioxidant hesperetin improves the quality of porcine oocytes during aging in vitro.

The citrus flavonoid hesperetin has a variety of pharmacological actions, including antioxidant, antiinflammatory, and anticancer activities. This study investigated whether hesperetin prevents aging of oocytes in vitro in which it determined the maturation of nuclear and cytoplasm and the developmental capacity of embryo by modulating the reactive oxygen species (ROS) level. Porcine oocytes were matured in vitro for 44 hr (control) and for an additional 24 hr in the presence of 0, 1, 10, 100, and 250 µM hesperetin (aging, H-1, H-10, H-100, and H-250, respectively). Although there was no difference in the rate of maturation among all the groups, both the control and H-100 groups significantly increased in the rate of cleavage and blastocyst formation compared to the aging group. The H-100 group significantly decreased ROS activity and increases the level of glutathione (GSH) and expression of the antioxidant genes (PRDX5, NFE2L, SOD1, and SOD2) compared with the aging group. The H-100 groups prevented aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase and increased the messenger RNA expression of cytoplasmic maturation factor genes (GDF9, CCNB1, BMP15, and MOS). Subsequently, both the control and H-100 groups significantly increased the total cell number and decreased the apoptosis cells at the blastocyst stage compared with aging group. The results indicate that hesperetin improves the quality of porcine oocytes by protecting them against oxidative stress during aging in vitro.

Antioxidant ß-cryptoxanthin enhances porcine oocyte maturation and subsequent embryo development in vitro.

Oxidative stress is partly responsible for the poor quality of IVM oocytes. The present study investigated the effects of the antioxidant ß-cryptoxanthin on the IVM of porcine oocytes and the in vitro development of the ensuing embryos. Oocytes were matured in IVM medium containing different concentrations of ß-cryptoxanthin (0, 0.1, 1, 10 or 100µM). Treatment with 1µM ß-cryptoxanthin (Group 1B) improved polar body extrusion and the expression of maturation-related genes in cumulus cells and oocytes compared with control. In addition, levels of reactive oxygen species decreased significantly in Group 1B, whereas there were significant increases in glutathione levels and expression of the antioxidant genes superoxide dismutase 1 and peroxiredoxin 5 in this group. After parthenogenetic activation, although the cleavage rate did not differ between the control and 1B groups, the blastocyst formation rate was higher in the latter. Moreover, the total number of cells per blastocyst and relative mRNA levels of pluripotency marker and antioxidant genes were significantly higher in the 1B compared with control group. These results demonstrate that ß-cryptoxanthin decreases oxidative stress in porcine oocytes and improves their quality and developmental potential.

Lysophosphatidic acid accelerates development of porcine embryos by activating formation of the blastocoel.

Culture media modifications, including the addition of various factors, are important for the in vitro production of oocytes and embryos. In this study, we investigated the effects of lysophosphatidic acid (LPA) on porcine embryo development. Porcine parthenogenetic embryos were cultured with 0, 0.1, 1, and 10 µM LPA for 7 days, or cultured in basic medium until Day 4 and then treated with LPA from Days 4 to 7. No difference in the in vitro development of embryos cultured with LPA for 7 days was observed. Conversely, rates of blastocyst and over-expanded blastocyst formation were higher in the 0.1 and 1 µM LPA-treated versus the other groups of embryos treated from Days 4 to 7. Moreover, formation of early blastocysts occurred earlier and embryo size was larger in LPA-treated compared to control embryos. Expression of Connexin 43 and gap junction and cell adhesion-related genes (GJC1 and CDH1, respectively) was also higher in LPA-treated compared to control embryos. Despite no difference in the blastocyst total cell number between groups, the apoptotic index was lower in the LPA-treated group than in the control group; indeed, BCL2L1 (B-cell lymphoma 2-like protein 1) expression increased while BAK (Bcl-2 homologous antagonist killer) decreased in the LPA-treated group. Thus, addition of LPA to the medium from Days 4 to 7 of culture improves blastocyst formation and aids the development of preimplantation embryos.