Loss of protein phosphatase 2A regulatory subunit PPP2R5A is associated with increased incidence of stress-induced proarrhythmia.

Background: Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme that controls Ca2+ homeostasis and contractility of the heart via dephosphorylation of regulatory proteins. In some genetically modified mouse models with increased arrhythmogenicity, a reduced expression of the regulatory subunit B56α of PP2A was found as a concomitant effect. Whether there is a general correlation between the abundance of B56α and the promotion of cardiac arrhythmogenesis remains unclear. Methods: The aim of this study was therefore to investigate the role of PP2A-B56α in the propensity for arrhythmic activity in the heart. The experimental analysis of this question has been addressed by using a mouse model with deletion of the PP2A-B56α gene, PPP2R5A (KO), in comparison to wild-type animals (WT). Evidence for arrhythmogenicity was investigated in whole animal, isolated heart and cardiomyocytes by ECG, recording of monophasic action potential (MAP) induced by programmed electrical stimulation (PES), measurement of Ca2+ transients under increased pacing frequencies and determination of total K+ channel currents (I K). Results: ECG measurements showed a prolongation of QT time in KO vs. WT. KO mice exhibited a higher rate of premature ventricular contractions in the ECG. MAP measurements in Langendorff-perfused KO hearts showed increased episodes of ventricular tachyarrhythmia induced by PES. However, the KO hearts showed values for MAP duration that were similar to those in WT hearts. In contrast, KO showed more myocardial cells with spontaneous arrhythmogenic Ca2+ transient events compared to WT. The whole-cell patch-clamp technique applied to ventricular cardiomyocytes revealed comparable peak potassium channel current densities between KO and WT. Conclusion: These findings support the assumption that a decrease or even the loss of PP2A-B56α leads to an increased propensity of triggered arrhythmias. This could be based on the increased spontaneous Ca2+ tansients observed.

Impaired myocellular Ca2+ cycling in protein phosphatase PP2A-B56α KO mice is normalized by ß-adrenergic stimulation.

The activity of protein phosphatase 2A (PP2A) is determined by the expression and localization of the regulatory B-subunits. PP2A-B56α is the dominant isoform of the B'-family in the heart. Its role in regulating the cardiac response to ß-adrenergic stimulation is not yet fully understood. We therefore generated mice deficient in B56α to test the functional cardiac effects in response to catecholamine administration versus corresponding WT mice. We found the decrease in basal PP2A activity in hearts of KO mice was accompanied by a counter-regulatory increase in the expression of B' subunits (ß and γ) and higher phosphorylation of sarcoplasmic reticulum Ca2+ regulatory and myofilament proteins. The higher phosphorylation levels were associated with enhanced intraventricular pressure and relaxation in catheterized KO mice. In contrast, at the cellular level, we detected depressed Ca2+ transient and sarcomere shortening parameters in KO mice at basal conditions. Consistently, the peak amplitude of the L-type Ca2+ current was reduced and the inactivation kinetics of ICaL were prolonged in KO cardiomyocytes. However, we show ß-adrenergic stimulation resulted in a comparable peak amplitude of Ca2+ transients and myocellular contraction between KO and WT cardiomyocytes. Therefore, we propose higher isoprenaline-induced Ca2+ spark frequencies might facilitate the normalized Ca2+ signaling in KO cardiomyocytes. In addition, the application of isoprenaline was associated with unchanged L-type Ca2+ current parameters between both groups. Our data suggest an important influence of PP2A-B56α on the regulation of Ca2+ signaling and contractility in response to ß-adrenergic stimulation in the myocardium.

Effects of estrogens and antiestrogens on gonadal sex differentiation and embryonic development in the domestic fowl (Gallus gallus domesticus).

Since it is known that environmental contaminants have the potential to cause endocrine disorders in humans and animals, there is an urgent need for in vivo tests to assess possible effects of these endocrine disrupting chemicals (EDCs). Although there is no standardized guideline, the avian embryo has proven to be particularly promising as it responds sensitively to a number of EDCs preferentially impacting the reproductive axis. In the present study we examined the effects of in ovo exposure to fulvestrant and tamoxifen as antiestrogenic model compounds and co-exposure to both substances and the potent estrogen 17α-ethinylestradiol (EE2) regarding sex differentiation and embryonic development of the domestic fowl (Gallus gallus domesticus). The substances were injected into the yolk of fertilized eggs on embryonic day 1. On embryonic day 19 sex genotype and phenotype were determined, followed by gross morphological and histological examination of the gonads. Sole EE2-treatment (20 ng/g egg) particularly affected male gonads and resulted in an increased formation of female-like gonadal cortex tissue and a reduction of seminiferous tubules. In ovo exposure to tamoxifen (0.1/1/10 µg/g egg) strongly impaired the differentiation of female gonads, led to a significant size reduction of the left ovary and induced malformations of the ovarian cortex, while fulvestrant (0.1/1/10 µg/g egg) did not affect sexual differentiation. However, both antiestrogens were able to antagonize the feminizing effects of EE2in genetic males when administered simultaneously. Since both estrogens and antiestrogens induce concentration-dependent morphological alterations of the sex organs, the chick embryo can be regarded as a promising model for the identification of chemicals with estrogenic and antiestrogenic activity.