BACKGROUND:It is unclear whether the increase in protein expression of PP2A regulatory subunit PR72 seen in human heart failure represents a primary compensatory mechanism or the final reaction to contractile decompensation. To address this question, we have explored the effects of chronic catecholaminergic stress in a transgenic (TG) mouse model with heart-specific overexpression of PR72 that exhibits hypercontractility at basal conditions. METHODS:Mice were treated with isoprenaline (ISO) or NaCl for 7 days using osmotic minipumps. Hearts or isolated cardiomyocytes from the animals were functionally examined. RESULTS:We could show (i) that PR72 expression is not only increased after chronic ISO stimulation but also in other different stress and insufficiency models. In TG mice, 7 days of ISO treatment led to (ii) increased hypertrophy, pulmonary edema, more fibrosis, and higher ACTA1 gene expression compared to wild-type (WT) mice. These effects were accompanied by (iii) a decrease in myocellular contractility and prolonged relaxation. Ca2+ transients (iv) showed correspondingly delayed decay kinetics in TG versus WT, while (v) the reduction of L-type calcium peak current by ISO treatment was less pronounced in TG cells. The decrease in RyR2 phosphorylation in TG (vi) supports a deterioration in contractility due to chronic ISO treatment in TG. CONCLUSION:Our results indicate that the upregulation of PP2A-PR72 in various stress and heart failure models has a long-term effect, perpetuating the molecular and functional detrimental cardiac changes, if it does not have a triggering effect.

Sphingosine 1-phosphate (S1P) is a lysosphingolipid with antiatherogenic properties, but mechanisms underlying its effects remain unclear. We here investigated atherosclerosis development in cholesterol-rich diet-fed LDL receptor-deficient mice with high or low overexpression levels of S1P receptor1 (S1P1) in macrophages. S1P1-overexpressing macrophages showed increased activity of transcription factors PU.1, interferon regulatory factor 8 (IRF8), and liverX receptor (LXR) and were skewed toward an M2-distinct phenotype characterized by enhanced production of IL-10, IL-1RA, and IL-5; increased ATP-binding cassette transporter A1- and G1-dependent cholesterol efflux; increased expression of MerTK and efferocytosis; and reduced apoptosis due to elevated B cell lymphoma 6 and Maf bZIP B. A similar macrophage phenotype was observed in mice administered S1P1-selective agonist KRP203. Mechanistically, the enhanced PU.1, IRF8, and LXR activity in S1P1-overexpressing macrophages led to downregulation of the cAMPdependent PKA and activation of the signaling cascade encompassing protein kinases AKT and mTOR complex 1 as well as the late endosomal/lysosomal adaptor MAPK and mTOR activator 1. Atherosclerotic lesions in aortic roots and brachiocephalic arteries were profoundly or moderately reduced in mice with high and low S1P1 overexpression in macrophages, respectively. We conclude that S1P1 signaling polarizes macrophages toward an antiatherogenic functional phenotype and countervails the development of atherosclerosis in mice.

Cardiomyocyte maturation during pre- and postnatal development requires multiple intertwined processes, including a switch in energy generation from glucose utilization in the embryonic heart towards fatty acid oxidation after birth. This is accompanied by a boost in mitochondrial mass to increase capacities for oxidative phosphorylation and ATP generation required for efficient contraction. Whether cardiomyocyte differentiation is paralleled by augmented capacities to deal with reactive oxygen species (ROS), physiological byproducts of the mitochondrial electron transport chain (ETC), is less clear. Here we show that expression of genes and proteins involved in redox homeostasis and protein quality control within mitochondria increases after birth in the mouse and human heart. Using primary embryonic, neonatal and adult mouse cardiomyocytes in vitro we investigated how excessive ROS production induced by mitochondrial dysfunction affects cell survival and stress response at different stages of maturation. Embryonic and neonatal cardiomyocytes largely tolerate inhibition of ETC complex III by antimycin A (AMA) as well as ATP synthase (complex V) by oligomycin but are susceptible to complex I inhibition by rotenone. All three inhibitors alter the intracellular distribution and ultrastructure of mitochondria in neonatal cardiomyocytes. In contrast, adult cardiomyocytes treated with AMA undergo rapid morphological changes and cellular disintegration. At the molecular level embryonic cardiomyocytes activate antioxidative defense mechanisms, the integrated stress response (ISR) and ER stress but not the mitochondrial unfolded protein response upon complex III inhibition. In contrast, adult cardiomyocytes fail to activate the ISR and antioxidative proteins following AMA treatment. In conclusion, our results identified fundamental differences in cell survival and stress response in differentiated compared to immature cardiomyocytes subjected to mitochondrial dysfunction. The high stress tolerance of immature cardiomyocytes might allow outlasting unfavorable intrauterine conditions thereby preventing fetal or perinatal heart disease and may contribute to the regenerative capacity of the embryonic and neonatal mammalian heart.

Abstract ID 128553Poster Board 072Introduction: Protein phosphatase 2A (PP2A) regulates cellular functions by dephosphorylation of target proteins. Dysregulation of PP2A is implicated in various diseases, making small molecules activating PP2A (SMAPs) a promising therapeutic approach. However, the specific role of SMAPs in individual tissues and the importance of specific regulatory B subunits of PP2A for activation remain to be determined.Objective: For this purpose, we investigated the effect of the SMAPs DT-061 and FTY720 (Fingolimod) on PP2A function in intact murine aortae, with regard to the role of the regulatory subunit PP2A-B56α, which has been proposed to be stabilized by DT-061.Materials & methods: Animal research was conducted in accordance with local animal welfare authorities and in compliance with the European Parliament’s Directive 2010/63/EU on the protection of animals used for scientific purposes. Levels of PP2A subunits were analyzed using quantitative RT-PCR. Aortic ring contractility was determined in wild-type (WT) and B56α knockout (KO) mice using wire myograph measurements. Aortic rings were incubated of FTY720 or DT-061, precontracted with phenylephrine (1 μM) and relaxation was analyzed by increasing isoproterenol (ISO) concentrations vs. appropriate controls.Results: Quantitative RT-PCR showed significant mRNA levels of almost all regulatory PP2A subunits in mouse aortic media, with the B56α subunit being highly represented (n = 8). Activation of PP2A with the PP2A activator DT-061 showed a concentration-dependent increase in relaxation in WT aortae compared to DMSO controls after 20 min of stimulation (DT-061: 10 μM 20 ± 7%, 30 μM 51 ± 14%, n = 4). Aortic rings stimulated with DT-061 (10 μM) showed a comparable left shift in dose-response curves of ISO-induced relaxation in WT and KO aorta vs. DMSO controls (n = 6). Interestingly, activation of PP2A with the PP2A activator FTY720 (5 μM) resulted in enhanced relaxation at increasing ISO concentrations in KO (IC50: 1.1 ± 0.4 μM) vs. WT (IC50: 5.6 ± 3.5 μM) aortic rings (n = 5). This effect was reversed by specific inhibition of PP2A in KO aortic rings with 3 nmol/L okadaic acid (+ 5 μM FTY720, n = 5).Conclusion: Our study reveals that PP2A activation by DT-061 enhances relaxation in ISO-stimulated aortae. In contrast, activation by FTY720 enhances ISO-induced relaxation only in the absence of the regulatory subunit B56α via PP2A-dependent mechanisms. This indicates that the B56α regulatory subunit is involved in regulating PP2A activation in vascular contractility. Notably, the activation of PP2A by FTY720 seems to be influenced by the presence of the B56α subunit, in contrast to DT-061, which was previously reported to target PP2A-B56α.

BackgroundThe activity, localization, and substrate specificity of the protein phosphatase 2A (PP2A) heterotrimer are controlled by various regulatory B subunits. PR72 belongs to the B'' gene family and has been shown to be upregulated in human heart failure. However, little is known about the functions of PR72 in the myocardium.MethodsTo address this issue, we generated a transgenic mouse model with heart-specific overexpression of PP2A-PR72. Biochemical and physiological methods were used to determine contractility, Ca2+ cycling parameters, and protein phosphorylation.ResultsA 2.5-fold increase in PR72 expression resulted in moderate cardiac hypertrophy. Maximal ventricular pressure was increased in catheterized transgenic mice (TG) compared to wild-type (WT) littermates. This was accompanied by an increased shortening of sarcomere length and faster relaxation at the single-cell level in TG. In parallel with these findings, the peak amplitude of Ca2+ transients was increased, and the decay in intracellular Ca2+ levels was shortened in TG compared to WT. The changes in Ca2+ cycling in TG were also evident from an increase in the full duration and width at half maximum of Ca2+ sparks. Consistent with the contractile data, phosphorylation of phospholamban at threonine-17 was higher in TG hearts. The lower expression of the Na+/Ca2+ exchanger may also contribute to the hypercontractile state in transgenic myocardium.ConclusionOur results suggest that PP2A-PR72 plays an important role in regulating cardiac contractile function and Ca2+ cycling, indicating that the upregulation of PR72 in heart failure is an attempt to compensate functionally.

The activity of protein phosphatase 2A (PP2A) is determined by the expression and localization of the regulatory B-subunits. PP2A-B56 alpha is the dominant isoform of the B'-family in the heart. Its role in regulating the cardiac response to beta-adrenergic stimulation is not yet fully understood. We therefore generated mice deficient in B56 alpha 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 (beta and gamma) 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 I-CaL were prolonged in KO cardiomyocytes. However, we show beta-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-B56 alpha on the regulation of Ca2+ signaling and contractility in response to beta-adrenergic stimulation in the myocardium.

Question:The angiotensin II (type 1) (AT1) receptor blocker (ARB) telmisartan (TEL) is beneficial for the treatment of individuals suffering from metabolic syndrome.As we have recently identified that the anti-obese action of TEL is attributed to dietindependent alterations in gut microbiota, we investigated here whether TEL influences gut barrier function.Methods: C57BL/6N mice were fed for 3 months with chow or high-fat diet (HFD) and treated with vehicle or TEL (8 mg/kg/day).Mucus thickness was determined by immunohistochemistry. Periodic acid-Schiff staining allowed the number of goblet cells to be counted.Using western blots, qPCR, and immunohistochemistry, factors related to mucus biosynthesis (Muc2, St6galnac), proliferation (Ki-67), or necroptosis (Rip3) were measured.The influence of the ARB losartan on cell viability was determined by using the mucus-producing cell line HT29MTX or small intestinal epithelial cells to investigate cell viability as solubility of TEL was too low for in-vitro experiments. Results:In response to HFD, mice developed obesity as well as leptin and insulin resistance, which were prevented by chronic TEL treatment.While mucus thickness was lower, Muc2 expression was only tendentially reduced in response to HFD feeding, and albumin excretion (as a sign of worsened intestinal permeability) was unchanged.Independent of feeding, TEL additionally reduced mucus thickness.Numbers of goblet cells were not affected by HFD feeding and TEL.St6galnac expression was increased by TEL.Rip3 was increased in TEL-treated and HFD-fed mice, while Ki-67 decreased.Cell viability was diminished by using >1 mM losartan. Conclusions:The anti-obese effect of TEL was associated with a decrease in mucus thickness, which was likely not related to a lower expression of Muc2 and goblet cells.A decrease in Ki-67 and increase in Rip3 indicates lower cell proliferation and necroptosis upon TEL.However, direct cell toxic effects are ruled out, as in-vivo concentrations are lower than 1 mM.This conclusion seems justified, as albumin excretion was not impaired in TEL-treated animals in our study.Nevertheless, followup studies are need to elucidate whether this detrimental TEL effect is relevant for gastrointestinal function.

ICER (inducible cAMP early repressor) isoforms are transcriptional repressors encoded by the Crem (cAMP responsive element modulator) gene. They were linked to the regulation of a multitude of cellular processes and pathophysiological mechanisms. Here, we show for the first time that two independent induction patterns for CREM repressor isoforms exist in the heart, namely for ICER and smICER (small ICER), which are induced in response to β-adrenergic stimulation in a transient- and saturation-like manner, respectively. This time-shifted induction pattern, driven by two internal promoters in the Crem gene, leads to the predominant transcription of smIcer after prolonged β-adrenergic stimulation. Using an ICER knockout mouse model with preserved smICER induction, we show that the transient-like induction of Icer itself has minor effects on gene regulation, cardiac hypertrophy or contractile function in the heart. We conclude that the functions previously linked to ICER may be rather attributed to smICER, also beyond the cardiac background.

BACKGROUND Transgenic mice (TG) with heart‐directed overexpresion of the isoform of the transcription factor cyclic adenosine monophosphate response element modulator (CREM), CREM‐IbΔC‐X, display spontaneous atrial fibrillation (AF) and action potential prolongation. The remodeling of the underlying ionic currents remains unknown. Here, we investigated the regulatory role of CREM‐IbΔC‐X on the expression of K+ channel subunits and the corresponding K+ currents in relation to AF onset in TG atrial myocytes. METHODS AND RESULTS ECG recordings documented the absence or presence of AF in 6‐week‐old (before AF onset) and 12‐week‐old TG (after AF onset) and wild‐type littermate mice before atria removal to perform patch clamp, contractility, and biochemical experiments. In TG atrial myocytes, we found reduced repolarization reserve K+ currents attributed to a decrease of transiently outward current and inward rectifier K+ current with phenotype progression, and of acetylcholine‐activated K+ current, age independent. The molecular determinants of these changes were lower mRNA levels of Kcnd2/3, Kcnip2, Kcnj2/4, and Kcnj3/5 and decreased protein levels of K+ channel interacting protein 2 (KChIP2 ), Kir2.1/3, and Kir3.1/4, respectively. After AF onset, inward rectifier K+ current contributed less to action potential repolarization, in line with the absence of outward current component, whereas the acetylcholine‐induced action potential shortening before AF onset (6‐week‐old TG mice) was smaller than in wild‐type and 12‐week‐old TG mice. Atrial force of contraction measured under combined vagal‐sympathetic stimulation revealed increased sensitivity to isoprenaline irrespective of AF onset in TG. Moreover, we identified Kcnd2, Kcnd3, Kcnj3, and Kcnh2 as novel CREM‐target genes. CONCLUSIONS Our study links the activation of cyclic adenosine monophosphate response element–mediated transcription to the proarrhythmogenic electrical remodeling of atrial inward rectifier K+ currents with a role in action potential duration, resting membrane stability, and vagal control of the electrical activity.

The authors regret the incorrect order of authors in the originally published manuscript. The authors would like to apologise for any inconvenience caused. Chronic β-adrenergic stimulation reverses depressed Ca handling in mice overexpressing inhibitor-2 of protein phosphatase 1Journal of Molecular and Cellular CardiologyVol. 125PreviewA higher expression/activity of type 1 serine/threonine protein phosphatase 1 (PP1) may contribute to dephosphorylation of cardiac regulatory proteins triggering the development of heart failure. Full-Text PDF