Efficacy of RyR2 inhibitor EL20 in induced pluripotent stem cell-derived cardiomyocytes from a patient with catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia syndrome that often leads to sudden cardiac death. The most common form of CPVT is caused by autosomal-dominant variants in the cardiac ryanodine receptor type-2 (RYR2) gene. Mutations in RYR2 promote calcium (Ca²⁺) leak from the sarcoplasmic reticulum (SR), triggering lethal arrhythmias. Recently, it was demonstrated that tetracaine derivative EL20 specifically inhibits mutant RyR2, normalizes Ca²⁺ handling and suppresses arrhythmias in a CPVT mouse model. The objective of this study was to determine whether EL20 normalizes SR Ca²⁺ handling and arrhythmic events in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a CPVT patient. Blood samples from a child carrying RyR2 variant RyR2 variant Arg-176-Glu (R176Q) and a mutation-negative relative were reprogrammed into iPSCs using a Sendai virus system. iPSC-CMs were derived using the Stemdiff^TM kit. Confocal Ca²⁺ imaging was used to quantify RyR2 activity in the absence and presence of EL20. iPSC-CMs harbouring the R176Q variant demonstrated spontaneous SR Ca²⁺ release events, whereas administration of EL20 diminished these abnormal events at low nanomolar concentrations (IC₅₀ = 82 nM). Importantly, treatment with EL20 did not have any adverse effects on systolic Ca²⁺ handling in control iPSC-CMs. Our results show for the first time that tetracaine derivative EL20 normalized SR Ca²⁺ handling and suppresses arrhythmogenic activity in iPSC-CMs derived from a CPVT patient. Hence, this study confirms that this RyR2-inhibitor represents a promising therapeutic candidate for treatment of CPVT.     

Regulation of cyclic guanosine monophosphate-dependent protein kinase in human uterine tissues during the menstrual cycle

Contractility of uterine smooth muscle is essential for the cyclic shedding of the endometrial lining and also for expulsion of the fetus during parturition. The nitric oxide (NO)-cGMP signaling pathway is involved in smooth muscle relaxation. The downstream target of this pathway essential for decreasing cytoplasmic calcium and muscle tone is the cGMP-dependent protein kinase (PKG). The present study was undertaken to localize expression of PKG in tissues of the female reproductive tract and to test the hypothesis that uterine smooth muscle PKG levels vary with the human menstrual cycle. Immunohistochemistry was used to localize PKG in myometrium, cervix, and endometrium obtained during proliferative and secretory phases. The PKG was localized to uterine and vascular smooth muscle cells in myometrium, stromal cells in endometrium, and a small percentage of cervical stromal cells. Using Western blot analysis and protein kinase activity assays, the expression of PKG was reduced significantly in progesterone-dominated uteri compared with myometrium from postmenopausal women or women in the proliferative phase. These findings support a role for PKG in the control of uterine and vascular smooth muscle contractility during the menstrual cycle.     

Age and gender affect DNMT3a and DNMT3b expression in human liver

DNA methylation is catalyzed by a family of DNA methyltransferases (DNMTs) including the maintenance enzyme DNMT 1 and de novo methyltransferases DNMT 3a and DNMT 3b. Elevated levels of DNMTs have been found in cancer cells and in several types of human tumors. A polymorphism found in DNMT3b has been associated with increased risk for several cancers. The factors influencing DNMT expression in human tissues have not been clearly determined. he present study examined TDNMT3a and DNMT3b levels in human liver tissue samples and compared the effect of ageing, cigarette smoking, and gender. DNMT3a and DNMT3b expression levels in the samples from older individuals (56–78 years, n = 28) were both significantly higher than those of the younger group (16–48 years, n = 27) (73.2 ± 3.4 vs 8.3 ± 2.8 and 56.1 ± 1.9 vs 17.5 ± 5.7, respectively; p < 0.05). Levels of DNMT3b in females were significantly higher than those in males (75.4 ± 2.2 vs 16.3 ± 4.7; p < 0.05); however, DNMT3a levels were similar for females and males (52.7 ± 2.7 vs 48.4 ± 2.0). Expression levels of DNMT3a and DNMT3b were similar in smokers and nonsmokers (58.1 ± 3.5 vs 60.8 ± 3.1 and 54.5 ± 2.3 vs 48.3 ± 1.8, respectively). Genotyping for DNMT3b (C→T) variant in this sample pool showed a frequency distribution of CC (41%), CT (50%), and TT (9%). The findings from this study suggest that ageing and gender may be important factors influencing DNA methylation status.     

Interior-Point Methods for Estimating Seasonal Parameters in Discrete-Time Infectious Disease Models

Infectious diseases remain a significant health concern around the world. Mathematical modeling of these diseases can help us understand their dynamics and develop more effective control strategies. In this work, we show the capabilities of interior-point methods and nonlinear programming (NLP) formulations to efficiently estimate parameters in multiple discrete-time disease models using measles case count data from three cities. These models include multiplicative measurement noise and incorporate seasonality into multiple model parameters. Our results show that nearly identical patterns are estimated even when assuming seasonality in different model parameters, and that these patterns show strong correlation to school term holidays across very different social settings and holiday schedules. We show that interior-point methods provide a fast and flexible approach to parameterizing models that can be an alternative to more computationally intensive methods.     

Pelvic Organ Support in Animals with Partial Loss of Fibulin-5 in the Vaginal Wall

Compromise of elastic fiber integrity in connective tissues of the pelvic floor is most likely acquired through aging, childbirth-associated injury, and genetic susceptibility. Mouse models of pelvic organ prolapse demonstrate systemic deficiencies in proteins that affect elastogenesis. Prolapse, however, does not occur until several months after birth and is thereby acquired with age or after parturition. To determine the impact of compromised levels of fibulin-5 (Fbln5) during adulthood on pelvic organ support after parturition and elastase-induced injury, tissue-specific conditional knockout (cKO) mice were generated in which doxycycline (dox) treatment results in deletion of Fbln5 in cells that utilize the smooth muscle α actin promoter-driven reverse tetracycline transactivator and tetracycline responsive element-Cre recombinase (i.e., Fbln5^f/f/SMA⁺⁺-rtTA/Cre⁺, cKO). Fbln5 was decreased significantly in the vagina of cKO mice compared with dox-treated wild type or controls (Fbln5^f/f/SMA⁺⁺-rtTA/Cre^-/-). In controls, perineal body length (PBL) and bulge increased significantly after delivery but declined to baseline values within 6–8 weeks. Although overt prolapse did not occur in cKO animals, these transient increases in PBL postpartum were amplified and, unlike controls, parturition-induced increases in PBL (and bulge) did not recover to baseline but remained significantly increased for 12 wks. This lack of recovery from parturition was associated with increased MMP-9 and nondetectable levels of Fbln5 in the postpartum vagina. This predisposition to prolapse was accentuated by injection of elastase into the vaginal wall in which overt prolapse occurred in cKO animals, but rarely in controls. Taken together, our model system in which Fbln5 is conditionally knock-downed in stromal cells of the pelvic floor results in animals that undergo normal elastogenesis during development but lose Fbln5 as adults. The results indicate that vaginal fibulin-5 during development is crucial for baseline pelvic organ support and is also important for protection and recovery from parturition- and elastase-induced prolapse.     

Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells.

Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca2+. Pretreatment of cells with 1-[N-O-bis(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca2+]i but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca2+ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca2+ and potentiated myosin light chain phosphorylation. We propose that the Ca(2+)-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca2+ sensitivity of myosin light chain phosphorylation in smooth muscle.