Paradoxical effect of anti-inflammatory drugs on IL-6 mRNA expression in patients with PTSD during treatment.

The pathophysiology of posttraumatic stress disorder (PTSD) is associated with the activation of the innate immune system, including cytokines like interleukin 6 (IL-6). However, the role of IL-6 in the etiology and treatment of PTSD still remains elusive. We conducted a prospective controlled trial to investigate the development of IL-6 during psychosomatic treatment in individuals with PTSD in comparison with individuals without PTSD. We assessed IL-6 mRNA expression before and after 2 months of psychosomatic treatment in individuals with and without PTSD. Severities of PTSD and depressive symptoms were assessed in parallel. Linear mixed regression was applied for statistical analysis, including the factors diagnosis PTSD and pre-post treatment after subgrouping for intake of anti-inflammatory drugs. The development of IL-6 mRNA expression during treatment was affected by the use of anti-inflammatory drugs. In the subgroup without intake of anti-inflammatory drugs, no significant statistical treatment effect in individuals with and without PTSD emerged. In the subgroup of individuals taking anti-inflammatory drugs, a significant interaction effect of the factors pre-post treatment and diagnosis PTSD was observed. Whereas IL-6 mRNA expression in individuals without PTSD decreased according to amelioration of symptoms, IL-6 mRNA expression in individuals with PTSD increased significantly during treatment, in opposite direction to symptom severity. Anti-inflammatory drugs might affect IL-6 mRNA expression in individuals with PTSD in a paradoxical way. This study offers a further piece of evidence that IL-6 could be involved in the pathophysiology of PTSD and PTSD-specific immunologic molecular mechanisms.

Endo Belly: What Is It and Why Does It Happen?-A Narrative Review.

Endometriosis is a chronic inflammatory disease where endometrial-like lesions settle outside the uterus, resulting in extensive inflammatory reactions. It is a complex disease that presents with a range of symptoms, with pain and infertility being the most common. Along with severe dysmenorrhea, cyclic and acyclic lower abdominal pain, cyclic dysuria and dyschezia, dyspareunia, and infertility, there are also nonspecific complaints that can cause confusion and make endometriosis the chameleon among gynecological diseases. These symptoms include unspecific intestinal complaints, cyclic diarrhea, but also constipation, nausea, vomiting, and stomach complaints. It appears that in addition to general bowel symptoms, there are also specific symptoms related to endometriosis such as cyclic bloating of the abdomen, known as endo belly. During the second half of the menstrual cycle leading up to menstruation, the abdomen becomes increasingly bloated causing discomfort and pain due to elevated sensitivity of the intestinal wall. Patients with endometriosis exhibit a reduced stretch pain threshold of the intestinal wall. Here, we review the endo belly, for the first time, pathophysiology and the influence of other diseases (such as irritable bowel syndrome-IBS), microbiome, hormonal levels, inflammation, and diet on the presentation of this condition.

Ablation of C-type natriuretic peptide/cGMP signaling in fibroblasts exacerbates adverse cardiac remodeling in mice.

Excessive activation of cardiac fibroblasts (CFs) in response to injury provokes cardiac fibrosis, stiffness, and failure. The local mediators counterregulating this response remain unclear. Exogenous C-type natriuretic peptide (CNP) exerts antifibrotic effects in preclinical models. To unravel the role of the endogenous hormone, we generated mice with fibroblast-restricted deletion (KO) of guanylyl cyclase-B (GC-B), the cGMP-synthesizing CNP receptor. CNP activated GC-B/cGMP signaling in human and murine CFs, preventing proliferative and promigratory effects of angiotensin II (Ang II) and TGF-ß. Fibroblast-specific GC-B-KO mice showed enhanced fibrosis in response to Ang II infusions. Moreover, after 2 weeks of mild pressure overload induced by transverse aortic constriction (TAC), such KO mice had augmented cardiac fibrosis and hypertrophy, together with systolic and diastolic contractile dysfunction. This was associated with increased expression of the profibrotic genes encoding collagen I, III, and periostin. Notably, such responses to Ang II and TAC were greater in female as compared with male KO mice. Enhanced Ang II-induced CNP expression in female hearts and augmented GC-B expression and activity in female CFs may contribute to this sex disparity. The results show that paracrine CNP signaling in CFs has antifibrotic and antihypertrophic effects. The CNP/GC-B/cGMP pathway might be a target for therapies combating pathological cardiac remodeling.

NK cells with tissue-resident traits shape response to immunotherapy by inducing adaptive antitumor immunity.

T cell-directed cancer immunotherapy often fails to generate lasting tumor control. Harnessing additional effectors of the immune response against tumors may strengthen the clinical benefit of immunotherapies. Here, we demonstrate that therapeutic targeting of the interferon-γ (IFN-γ)-interleukin-12 (IL-12) pathway relies on the ability of a population of natural killer (NK) cells with tissue-resident traits to orchestrate an antitumor microenvironment. In particular, we used an engineered adenoviral platform as a tool for intratumoral IL-12 immunotherapy (AdV5-IL-12) to generate adaptive antitumor immunity. Mechanistically, we demonstrate that AdV5-IL-12 is capable of inducing the expression of CC-chemokine ligand 5 (CCL5) in CD49a+ NK cells both in tumor mouse models and tumor specimens from patients with cancer. AdV5-IL-12 imposed CCL5-induced type I conventional dendritic cell (cDC1) infiltration and thus increased DC-CD8 T cell interactions. A similar observation was made for other IFN-γ-inducing therapies such as Programmed cell death 1 (PD-1) blockade. Conversely, failure to respond to IL-12 and PD-1 blockade in tumor models with low CD49a+ CXCR6+ NK cell infiltration could be overcome by intratumoral delivery of CCL5. Thus, therapeutic efficacy depends on the abundance of NK cells with tissue-resident traits and, specifically, their capacity to produce the DC chemoattractant CCL5. Our findings reveal a barrier for T cell-focused therapies and offer mechanistic insights into how T cell-NK cell-DC cross-talk can be enhanced to promote antitumor immunity and overcome resistance.

Analyzing Longitudinal Data on Singing Voice Parameters of Boys and Girls Aged 8 to 12.5 and Possible Effects of a Music Pedagogical Intervention.

OBJECTIVE: Controlled and randomized study to analyze longitudinal voice data of boys and girls aged 8-12.5, to describe their physiological development and to evaluate the influence of a one-year music pedagogical interventional program focused on the singing voice. METHODS: Singing voice profiles of 116 children (52 boys, 64 girls) aged 8-12.5 years were collected longitudinally at the beginning and the end of the third school year and the end of the fourth school year. 64 of the 116 children received a music pedagogical program during their third school year (interventional group). Maximum and minimum voice intensity, highest and lowest frequency, maximum phonation time (MPT) and Jitter were investigated. RESULTS: In two years' time highest frequency increased on average by 100.23 Hz from G1 up to A1 for boys and for girls. Lowest frequency decreased by 18.36Hz from Gis-1 to G-1 (boys: Gis-1-Fis-1; girls: A-1-G-1). There was no clinically relevant development of the intensity parameters for both sexes. However, after the interventional year, minimum voice intensity significantly decreased in the interventional group compared to the control group. The MPT increased by 2.41 seconds from 10.67 seconds up to 13.09 seconds in two years. Here the increase was higher for boys (+3.2 seconds) than for girls (+1.77 seconds). Jitter was found to be 0.84%-1.11%, showing no clinically relevant changes in two years. CONCLUSION: To the best of our knowledge, for the first time this study presents longitudinal data on singing voice parameters of the voice range profile of boys and girls aged 8-12.5 years. While frequency and intensity parameters develop equally for boys and girls, the MPT is found to develop more distinctly within boys. A music pedagogical intervention of a small extent has a positive effect on the ability to sing as soft as possible.

Fluorescence-encoded poly(methyl metharcylate) nanoparticles for a lateral flow assay detecting IgM autoantibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) belongs to the most often occurring autoimmune diseases in the world. For serological diagnosis, IgM auto-antibodies directed against the Fc portion of IgG referred to as rheumatoid factor are used as biomarkers. The autoantibody detection is usually done by ELISA. Such assays are reliable but are not suitable for point-of-care testing in contrast to lateral flow assays. Here, we report the development of a lateral flow assay based on carboxylated fluorescence-encoded poly(methyl methacrylate) nanoparticles. Poly(methyl methacrylate) is a non-toxic plastic with an excellent biocompatibility and high optical transparency which promises especially high sensitive fluorescence detection thereby leading to very sensitive assays. We could detect a positive signal in samples with a nephelometric reading down to 0.4 U/mL. By analyzing 30 sera of patients with a RA diagnosis and 34 sera of healthy test subjects we could confirm positive ELISA results in 72% of all cases and negative ELISA results in 97% of all cases.

Loss of Lymphotoxin Alpha-Expressing Memory B Cells Correlates with Metastasis of Human Primary Melanoma.

Activated antigen-experienced B cells play an unexpected complex role in anti-tumor immunity in human melanoma patients. However, correlative studies between B cell infiltration and tumor progression are limited by the lack of distinction between functional B cell subtypes. In this study, we examined a series of 59 primary and metastatic human cutaneous melanoma specimens with B cell infiltration. Using seven-color multiplex immunohistochemistry and automated tissue imaging and analysis, we analyzed the spatiotemporal dynamics of three major antigen-experienced B cell subpopulations expressing lymphotoxin alpha (LTA/TNFSF1) or interleukin-10 (IL-10) outside tertiary lymphoid structures. The expression of both LTA and IL-10 was not restricted to a particular B cell subtype. In primary melanomas, these cells were predominantly found at the invasive tumor-stroma front and, in metastatic melanomas, they were also found in the intratumoral stroma. In primary melanomas, decreased densities of LTA+ memory-like and, to a lesser extent, activated B cells were associated with metastasis. Compared with metastatic primary tumors, B cell infiltrates in melanoma metastases were enriched in both LTA+ memory-like and LTA+ activated B cells, but not in any of the IL-10+ B cell subpopulations. Melanoma disease progression shows distinct dynamics of functional B cell subpopulations, with the regulation of LTA+ B cell numbers being more significant than IL-10+ B cell subpopulations.

A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration.

There is increasing evidence that tertiary lymphoid structures (TLS) control not only local adaptive B cell responses at melanoma tumor sites but also the cellular composition and function of other immune cells. In human melanoma, however, a comprehensive analysis of TLS phenotypes, density and spatial distribution at different disease stages is lacking. Here we used 7-color multiplex immunostaining of whole tissue sections from 103 human melanoma samples to characterize TLS phenotypes along the expression of established TLS-defining molecular and cellular components. TLS density and spatial distribution were determined by referring TLS counts to the tissue area within defined intra- and extratumoral perimeters around the invasive tumor front. We show that only a subgroup of primary human melanomas contains TLS. These TLS rarely formed germinal centers and mostly located intratumorally within 1 mm distance to the invasive tumor front. In contrast, melanoma metastases had a significantly increased density of secondary follicular TLS. They appeared preferentially in stromal areas within an extratumoral 1 mm distance to the invasive tumor front and their density varied over time and site of metastasis. Interestingly, secondary follicular TLS in melanoma often lacked BCL6+ lymphatic cells and canonical germinal center polarity with the formation of dark and light zone areas. Our work provides an integrated qualitative, quantitative and spatial analysis of TLS in human melanoma and shows disease progression- and site-associated changes in TLS phenotypes, density and spatial distribution. The frequent absence of canonical germinal center polarity in melanoma TLS highlights the induction of TLS maturation as a potential additive to future immunotherapy studies. Given the variable evaluation strategies used in previous TLS studies of human tumors, an important asset of this study is the standardized quantitative evaluation approach that provides a high degree of reproducibility.

Spatiotemporal Analysis of B Cell- and Antibody Secreting Cell-Subsets in Human Melanoma Reveals Metastasis-, Tumor Stage-, and Age-Associated Dynamics.

Background: The role of tumor-associated B cells in human cancer is only starting to emerge. B cells typically undergo a series of developmental changes in phenotype and function, however, data on the composition of the B cell population in human melanoma are largely absent including changes during tumor progression and their potential clinical significance. Methods: In this study, we compared the number and distribution of six major B cell and antibody secreting cell subpopulations outside tertiary lymphoid structures in whole tumor sections of 154 human cutaneous melanoma samples (53 primary tumors without subsequent metastasis, 44 primary tumors with metastasis, 57 metastatic samples) obtained by seven color multiplex immunohistochemistry and automated tissue imaging and analysis. Results: In primary melanomas, we observed the highest numbers for plasmablast-like, memory-like, and activated B cell subtypes. These cells showed a patchy, predominant paratumoral distribution at the invasive tumor-stroma margin. Plasma cell-like cells were hardly detected, germinal center- and transitional/regulatory-like B cells not at all. Of the major clinicopathologic prognostic factors for primary melanomas, metastasis was associated with decreased memory-like B cell numbers and a higher age associated with higher plasmablast-like cell numbers. When we compared the composition of B cell subpopulations in primary melanomas and metastatic samples, we found a significantly higher proportion of plasma cell-like cells at distant metastatic sites and a higher proportion of memory-like B cells at locoregional than distant metastatic sites. Both cell types were detected mainly in the para- and intratumoral stroma. Conclusion: These data provide a first comprehensive and comparative spatiotemporal analysis of major B cell and antibody secreting cell subpopulations in human melanoma and describe metastasis-, tumor stage-, and age-associated dynamics, an important premise for B cell-related biomarker and therapy studies.

Simultaneous imaging of widely differing particle concentrations in MPI: problem statement and algorithmic proposal for improvement.

Magnetic particle imaging (MPI) is a tomographic imaging technique for determining the spatial distribution of superparamagnetic nanoparticles. Current MPI systems are capable of imaging iron masses over a wide dynamic range of more than four orders of magnitude. In theory, this range could be further increased using adaptive amplifiers, which prevent signal clipping. While this applies to a single sample, the dynamic range is severely limited if several samples with different concentrations or strongly inhomogeneous particle distributions are considered. One scenario that occurs quite frequently in pre-clinical applications is that a highly concentrated tracer bolus in the vascular system 'shadows' nearby organs with lower effective tracer concentrations. The root cause of the problem is the ill-posedness of the MPI imaging operator, which requires regularization for stable reconstruction. In this work, we introduce a simple two-step algorithm that increases the dynamic range by a factor of four. Furthermore, the algorithm enables spatially adaptive regularization, i.e. highly concentrated signals can be reconstructed with maximum spatial resolution, while low concentrated signals are strongly regularized to prevent noise amplification.

C-type natriuretic peptide moderates titin-based cardiomyocyte stiffness.

Heart failure is often accompanied by titin-dependent myocardial stiffness. Phosphorylation of titin by cGMP-dependent protein kinase I (PKGI) increases cardiomyocyte distensibility. The upstream pathways stimulating PKGI-mediated titin phosphorylation are unclear. We studied whether C-type natriuretic peptide (CNP), via its guanylyl cyclase-B (GC-B) receptor and cGMP/PKGI signaling, modulates titin-based ventricular compliance. To dissect GC-B-mediated effects of endogenous CNP in cardiomyocytes, we generated mice with cardiomyocyte-restricted GC-B deletion (CM GC-B-KO mice). The impact on heart morphology and function, myocyte passive tension, and titin isoform expression and phosphorylation was studied at baseline and after increased afterload induced by transverse aortic constriction (TAC). Pressure overload increased left ventricular endothelial CNP expression, with an early peak after 3 days. Concomitantly, titin phosphorylation at Ser4080, the site phosphorylated by PKGI, was augmented. Notably, in CM GC-B-KO mice this titin response was abolished. TAC-induced hypertrophy and fibrosis were not different between genotypes. However, the KO mice presented mild systolic and diastolic dysfunction together with myocyte stiffness, which were not observed in control littermates. In vitro, recombinant PKGI rescued reduced titin-Ser4080 phosphorylation and reverted passive stiffness of GC-B-deficient cardiomyocytes. CNP-induced activation of GC-B/cGMP/PKGI signaling in cardiomyocytes provides a protecting regulatory circuit preventing titin-based myocyte stiffening during early phases of pressure overload.

An Activating Deletion Variant in the Submembrane Region of Natriuretic Peptide Receptor-B Causes Tall Stature.

CONTEXT: C-type natriuretic peptide (CNP) is critically involved in endochondral bone growth. Variants in the genes encoding CNP or its cyclic guanosine monophosphate (cGMP)-forming receptor (natriuretic peptide receptor-B [NPR-B], gene NPR2) cause monogenic growth disorders. Here we describe a novel gain-of-function variant of NPR-B associated with tall stature and macrodactyly of the great toes (epiphyseal chondrodysplasia, Miura type). DESIGN: History and clinical characteristics of 3 family members were collected. NPR2 was selected for sequencing. Skin fibroblasts and transfected HEK-293 cells were used to compare mutant versus wild-type NPR-B activities. Homology modeling was applied to understand the molecular consequences of the variant. RESULTS: Mother's height was +2.77 standard deviation scores (SDS). The heights of her 2 daughters were +1.96 SDS at 7 years and +1.30 SDS at 4 years of age. Skeletal surveys showed macrodactyly of the great toes and pseudo-epiphyses of the mid- and proximal phalanges. Sequencing identified a novel heterozygous variant c.1444_1449delATGCTG in exon 8 of NPR2, predicted to result in deletion of 2 amino acids Met482-Leu483 within the submembrane region of NPR-B. In proband's skin fibroblasts, basal cGMP levels and CNP-stimulated cGMP production were markedly increased compared with controls. Consistently, assays with transfected HEK-293 cells showed markedly augmented baseline and ligand-dependent activity of mutant NPR-B. CONCLUSIONS: We report the second activating variant within the intracellular submembrane region of NPR-B resulting in tall stature and macrodactyly. Our functional and modeling studies suggest that this domain plays a critical role in the baseline conformation and ligand-dependent structural rearrangement of NPR-B required for cGMP production.

Stabilization of Perivascular Mast Cells by Endothelial CNP (C-Type Natriuretic Peptide).

OBJECTIVE: Activated perivascular mast cells (MCs) participate in different cardiovascular diseases. Many factors provoking MC degranulation have been described, while physiological counterregulators are barely known. Endothelial CNP (C-type natriuretic peptide) participates in the maintenance of vascular barrier integrity, but the target cells and mechanisms are unclear. Here, we studied whether MCs are regulated by CNP. Approach and Results: In cultured human and murine MCs, CNP activated its specific GC (guanylyl cyclase)-B receptor and cyclic GMP signaling. This enhanced cyclic GMP-dependent phosphorylation of the cytoskeleton-associated VASP (vasodilator-stimulated phosphoprotein) and inhibited ATP-evoked degranulation. To elucidate the relevance in vivo, mice with a floxed GC-B (Npr2) gene were interbred with a Mcpt5-CreTG line to generate mice lacking GC-B in connective tissue MCs (MC GC-B knockout). In anesthetized mice, acute ischemia-reperfusion of the cremaster muscle microcirculation provoked extensive MC degranulation and macromolecule extravasation. Superfusion of CNP markedly prevented MC activation and endothelial barrier disruption in control but not in MC GC-B knockout mice. Notably, already under resting conditions, such knockout mice had increased numbers of degranulated MCs in different tissues, together with elevated plasma chymase levels. After transient coronary occlusion, their myocardial areas at risk and with infarction were enlarged. Moreover, MC GC-B knockout mice showed augmented perivascular neutrophil infiltration and deep vein thrombosis in a model of inferior vena cava ligation. CONCLUSIONS: CNP, via GC-B/cyclic GMP signaling, stabilizes resident perivascular MCs at baseline and prevents their excessive activation under pathological conditions. Thereby CNP contributes to the maintenance of vascular integrity in physiology and disease.

Natriuretic Peptides Attenuate Retinal Pathological Neovascularization Via Cyclic Guanosine Monophosphate Signaling in Pericytes and Astrocytes.

OBJECTIVE: In proliferative retinopathies, complications derived from neovascularization cause blindness. During early disease, pericyte's apoptosis contributes to endothelial dysfunction and leakage. Hypoxia then drives VEGF (vascular endothelial growth factor) secretion and pathological neoangiogenesis. Cardiac ANP (atrial natriuretic peptide) contributes to systemic microcirculatory homeostasis. ANP is also formed in the retina, with unclear functions. Here, we characterized whether endogenously formed ANP regulates retinal (neo)angiogenesis. Approach and Results: Retinal vascular development and ischemia-driven neovascularization were studied in mice with global deletion of GC-A (guanylyl cyclase-A), the cGMP (cyclic guanosine monophosphate)-forming ANP receptor. Mice with a floxed GC-A gene were interbred with Tie2-Cre, GFAP-Cre, or PDGF-Rß-CreERT2 lines to dissect the endothelial, astrocyte versus pericyte-mediated actions of ANP in vivo. In neonates with global GC-A deletion (KO), vascular development was mildly delayed. Moreover, such KO mice showed augmented vascular regression and exacerbated ischemia-driven neovascularization in the model of oxygen-induced retinopathy. Notably, absence of GC-A in endothelial cells did not impact retinal vascular development or pathological neovascularization. In vitro ANP/GC-A/cGMP signaling, via activation of cGMP-dependent protein kinase I, inhibited hypoxia-driven astrocyte's VEGF secretion and TGF-ß (transforming growth factor beta)-induced pericyte apoptosis. In neonates lacking ANP/GC-A signaling in astrocytes, vascular development and hyperoxia-driven vascular regression were unaltered; ischemia-induced neovascularization was modestly increased. Remarkably, inactivation of GC-A in pericytes retarded physiological retinal vascularization and markedly enhanced cell apoptosis, vascular regression, and subsequent neovascularization in oxygen-induced retinopathy. CONCLUSIONS: Protective pericyte effects of the ANP/GC-A/cGMP pathway counterregulate the initiation and progression of experimental proliferative retinopathy. Our observations indicate augmentation of endogenous pericyte ANP signaling as target for treatment of retinopathies associated with neovascularization.

Initial Characterization of Stressed Transgenic Mice With Cardiomyocyte-Specific Overexpression of Protein Phosphatase 2C.

As part of our ongoing studies on the potential pathophysiological role of serine/threonine phosphatases (PP) in the mammalian heart, we have generated mice with cardiac-specific overexpression of PP2Cß (PP2C-TG) and compared them with littermate wild type mice (WT) serving as a control. Cardiac fibrosis was noted histologically in PP2C-TG. Collagen 1a, interleukin-6 and the natriuretic peptides ANP and BNP were augmented in PP2C-TG vs. WT (p < 0.05). Left atrial preparations from PP2C-TG were less resistant to hypoxia than atria from WT. PP2C-TG maintained cardiac function after the injection of lipopolysaccharide (LPS, a model of sepsis) and chronic isoproterenol treatment (a model of heart failure) better than WT. Crossbreeding of PP2C-TG mice with PP2A-TG mice (a genetic model of heart failure) resulted in double transgenic (DT) mice that exhibited a pronounced increase of heart weight in contrast to the mild hypertrophy noted in the mono-transgenic mice. The ejection fraction was reduced in PP2C-TG and in PP2A-TG mice compared with WT, but the reduction was the highest in DT compared with WT. PP2A enzyme activity was enhanced in PP2A-TG and DT mice compared with WT and PP2C-TG mice. In summary, cardiac overexpression of PP2Cß and co-overexpression of both the catalytic subunit of PP2A and PP2Cß were detrimental to cardiac function. PP2Cß overexpression made cardiac preparations less resistant to hypoxia than WT, leading to fibrosis, but PP2Cß overexpression led to better adaptation to some stressors, such as LPS or chronic ß-adrenergic stimulation. Hence, the effect of PP2Cß is context sensitive.

Overexpression of protein phosphatase 5 in the mouse heart: Reduced contractility but increased stress tolerance - Two sides of the same coin?

The pathophysiological mechanisms of sepsis-induced cardiac dysfunction are largely unknown. The Toll-like receptor 4 (TLR4) is expressed in cardiac myocytes and is involved in bacterial endotoxin-mediated inflammatory disorders. TLR4 signaling leads to activation of the nuclear factor kappa B followed by increased expression of cytokines. Several protein phosphatases including PP2Cß, PP2A or PP1 are known to act as regulators of this signaling pathway. Here, we examined the role of PP5 for the inflammatory response to the bacterial endotoxin lipopolysaccharide in the heart using a transgenic mouse model with cardiac myocyte directed overexpression of PP5. In these transgenic mice, basal cardiac contractility was reduced, in vivo as well as in vitro, but LPS-induced cardiac dysfunction was less pronounced compared to wild type mice. Quantitative RT-PCR suggested an attenuated NF-κB signaling in the heart and cardiac expression of heat shock protein 25 (HSP25) was increased in PP5 transgenic mice. From our data we assume that PP5 increases stress tolerance of cardiac myocytes by downregulation of NF-κB signaling and upregulation of HSP25 expression.

SPRED2 deficiency elicits cardiac arrhythmias and premature death via impaired autophagy.

Cardiac functionality is dependent on a balanced protein turnover. Accordingly, regulated protein decay is critical to maintain cardiac function. Here we demonstrate that deficiency of SPRED2, an intracellular repressor of ERK-MAPK signaling markedly expressed in human heart, resulted in impaired autophagy, heart failure, and shortened lifespan. SPRED2-/- mice showed cardiomyocyte hypertrophy, cardiac fibrosis, impaired electrical excitability, and severe arrhythmias. Mechanistically, cardiomyocyte dysfunction resulted from ERK hyperactivation and dysregulated autophagy, observed as accumulation of vesicles, vacuolar structures, and degenerated mitochondria. The diminished autophagic flux in SPRED2-/- hearts was reflected by a reduced LC3-II/LC3-I ratio and by decreased Atg7, Atg4B and Atg16L expression. Furthermore, the autophagosomal adaptors p62/SQSTM1 and NBR1 and lysosomal Cathepsin D accumulated in SPRED2-/- hearts. In wild-type hearts, SPRED2 interacted physically with p62/SQSTM1, NBR1, and Cathepsin D, indicating that SPRED2 is required for autophagolysosome formation in regular autophagy. Restored inhibition of MAPK signaling by selumetinib led to an increase in autophagic flux in vivo. Therefore, our study identifies SPRED2 as a novel, indispensable regulator of cardiac autophagy. Vice versa, SPRED2 deficiency impairs autophagy, leading to cardiac dysfunction and life-threatening arrhythmias.

ß Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice.

BACKGROUND: The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and ß-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of ß-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in ß-cells. METHODS: Mice with a floxed GC-A gene were bred to Rip-CreTG mice, thereby deleting GC-A selectively in ß-cells (ß GC-A KO). Weight gain, glucose tolerance, insulin sensitivity, and glucose-stimulated insulin secretion were monitored in normal diet (ND)- and high-fat diet (HFD)-fed mice. ß-cell size and number were measured by immunofluorescence-based islet morphometry. RESULTS: In vitro, the insulinotropic and proliferative actions of ANP were abolished in islets isolated from ß GC-A KO mice. Concordantly, in vivo, infusion of BNP mildly enhanced baseline plasma insulin levels and glucose-induced insulin secretion in control mice. This effect of exogenous BNP was abolished in ß GC-A KO mice, corroborating the efficient inactivation of the GC-A receptor in ß-cells. Despite this under physiological, ND conditions, fasted and fed insulin levels, glucose-induced insulin secretion, glucose tolerance and ß-cell morphology were similar in ß GC-A KO mice and control littermates. However, HFD-fed ß GC-A KO animals had accelerated glucose intolerance and diminished adaptative ß-cell proliferation. CONCLUSIONS: Our studies of ß GC-A KO mice demonstrate that the cardiac hormones ANP and BNP do not modulate ß-cell's growth and secretory functions under physiological, normal dietary conditions. However, endogenous NP/GC-A signaling improves the initial adaptative response of ß-cells to HFD-induced obesity. Impaired ß-cell NP/GC-A signaling in obese individuals might contribute to the development of type 2 diabetes.