Phenotypic alterations in type II alveolar epithelial cells in CD4+ T cell mediated lung inflammation

<p>Abstract</p> <p>Background</p> <p>Although the contribution of alveolar type II epithelial cell (AEC II) activities in various aspects of respiratory immune regulation has become increasingly appreciated, our understanding of the contribution of AEC II transcriptosome in immunopathologic lung injury remains poorly understood. We have previously established a mouse model for chronic T cell-mediated pulmonary inflammation in which influenza hemagglutinin (HA) is expressed as a transgene in AEC II, in mice expressing a transgenic T cell receptor specific for a class II-restricted epitope of HA. Pulmonary inflammation in these mice occurs as a result of CD4⁺T cell recognition of alveolar antigen. This model was utilized to assess the profile of inflammatory mediators expressed by alveolar epithelial target cells triggered by antigen-specific recognition in CD4⁺T cell-mediated lung inflammation.</p> <p>Methods</p> <p>We established a method that allows the flow cytometric negative selection and isolation of primary AEC II of high viability and purity. Genome wide transcriptional profiling was performed on mRNA isolated from AEC II isolated from healthy mice and from mice with acute and chronic CD4⁺T cell-mediated pulmonary inflammation.</p> <p>Results</p> <p>T cell-mediated inflammation was associated with expression of a broad array of cytokine and chemokine genes by AEC II cell, indicating a potential contribution of epithelial-derived chemoattractants to the inflammatory cell parenchymal infiltration. Morphologically, there was an increase in the size of activated epithelial cells, and on the molecular level, comparative transcriptome analyses of AEC II from inflamed versus normal lungs provide a detailed characterization of the specific inflammatory genes expressed in AEC II induced in the context of CD4⁺T cell-mediated pneumonitis.</p> <p>Conclusion</p> <p>An important contribution of AEC II gene expression to the orchestration and regulation of interstitial pneumonitis is suggested by the panoply of inflammatory genes expressed by this cell population, and this may provide insight into the molecular pathogenesis of pulmonary inflammatory states. CD4⁺T cell recognition of antigen presented by AEC II cells appears to be a potent trigger for activation of the alveolar cell inflammatory transcriptosome.</p

The role of tachycardia and beta-adrenergic stimulation in inducing early cardiac remodelling

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): BioTechMed Graz Objective Cardiac remodelling encompasses changes at the molecular, cellular and gene expression level following pathologic insult to the heart. Initially, it maintains cardiovascular homeostasis and allows patients to remain asymptomatic, but if untreated, it eventually progresses to symptomatic heart failure. Excessive β-adrenergic stimulation and tachycardia are potent triggers of cardiac remodelling; however, the underlying mechanisms of their cellular effects are not fully understood. Using neonatal rat ventricular cardiac myocytes (NRVCMs), we studied individual and synergistic potency of ß-adrenergic stimulation and tachycardia to modulate pathological gene expression profiles, as well as the effectiveness of ß-blockers (BB) in preventing these alterations. Methods Primary NRVCMs were isolated from 1-day-old neonatal Wistar rats, cultured for 3 days and subsequently stimulated for 3h at basal (1Hz) and tachycardia (8Hz) conditions either in (1) cell culture medium to determine the sole effect of tachycardia, (2) cell culture medium supplemented with ß-adrenergic agonist isoprenaline (ISO; 10µM) to investigate the influence of ß-adrenergic stimulation and signalling or (3) cell culture medium supplemented with ISO following 1h preincubation with propranolol (ISO+BB; 1µM) to assess the potential of BB in preventing gene reprogramming. Screening of relative mRNA levels of hypertrophic marker genes and regulators of ion homeostasis in cardiomyocytes was performed by qPCR and calculated using the 2-ΔΔCt quantification method. Results qPCR screening of the known hypertrophic marker genes revealed that tachycardia caused significant transcriptional upregulation of regulator of calcineurin 1 (RCAN1) and interleukin-6 receptor (IL6R). Treatment with ISO additionally upregulated RCAN1, while preincubation with BB resulted in a return towards baseline expression of both genes, completely blocking the effects of tachycardia alone or when combined with ISO stimulation. Interestingly, two potassium channel genes, KCNH2 and KCNJ2, responsible for expression of hERG and Kir2.1 channels, respectively, were unchanged with tachycardia alone but significantly downregulated upon additional stimulation with ISO. Preincubation with BB could - at least partially - reverse the effect. Conclusion In conclusion, we could show that apart from the well-documented effect of excessive ß-adrenergic stimulation on hypertrophic signalling in cardiomyocytes, it also has a direct, non-tachypacing mediated effect on the expression levels of hERG and Kir2.1 potassium channels, which may be causally involved in inducing early cardiac remodelling. Thus, a previously unidentified benefit of BB therapy may be restoring potassium homeostasis contributing to the prevention of adverse cardiac remodelling and its progression to heart failure. </jats:sec

Beta-1 adrenergic receptor signalling during early and late hypertensive cardiac remodelling

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): BioTechMed Graz Objective Chronic activation of β1-adrenergic receptors (β1AR) in response to hypertension is consistently linked to maladaptive remodelling in the heart, however, the underlying mechanisms are not well understood. Here, our aim was to determine the subcellular profile and extent of β1AR expression at baseline and upon acute β-adrenergic stimulation in cardiac myocytes during early- and late-stage cardiac remodelling due to systemic hypertension. Methods Male Dahl salt-sensitive rats were fed a high-salt diet (HSD; 8% NaCl) for either five or ten weeks to induce early or late hypertensive cardiac remodelling, respectively. Age-, sex- and weight-matched Dahl salt-sensitive rats on a low-salt diet (LSD; 0.3% NaCl) served as controls. To test the effect of conventional anti-hypertensive treatment, a subset of HSD-fed animals received daily doses of the angiotensin-converting-enzyme-inhibitor Imidapril (ACE-I; 1mg/kg/day) starting two weeks after the feeding protocol was switched to HSD. Isolated ventricular myocytes were stimulated either under control conditions or in the presence of β-adrenergic agonist isoprenaline (ISO; 100nM; 1h). Confocal imaging of single cardiomyocytes allowed detailed quantification of β1AR in different cellular compartments. Finally, immunoblotting and microarray analyses were applied to quantify β1AR in the left ventricles of the corresponding groups of animals. Results In control rats, β1AR was found in a striated pattern throughout the cell typical for T-tubular network and in the perinuclear regions, while its expression significantly dropped upon ISO treatment. During early remodelling, basal β1AR expression was unchanged, but increased on the T-tubules and perinuclear regions upon acute stimulation with ISO. In contrast, late remodelling was marked by reduced β1AR expression at baseline, and significantly blunted increase in response to ISO compared to early time point. Interestingly, daily ACE-I treatment resulted in even more adverse phenotype as compared to untreated HSD-fed rats in early remodelling, but favourable control-like characteristics at late remodelling stage. Immunoblotting and microarrays from left ventricular tissue confirmed the data, where applicable. Conclusion Taken together, we showed that early hypertensive remodelling is marked by altered β1AR responsiveness upon β-adrenergic stimulation, whereas late remodelling also exhibits altered β1AR expression. ACE-I treatment seemed to interfere with early adaptive mechanisms, thereby worsening the phenotype as compared to untreated HSD-fed animals. However, upon prolonged application, it showed a clear protective effect from pathological molecular alterations at late remodelling. Further experiments involving downstream targets of β1AR signalling are required to fully understand the molecular sequence of events leading to early and late alterations in molecular composition of cardiomyocytes in the hypertensive heart. </jats:sec

Functional remodeling of perinuclear mitochondria alters nucleoplasmic Ca2+ signaling in heart failure

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): BioTechMed-Graz Mitochondrial dysfunction in cardiomyocytes is a hallmark of heart failure (HF) development. Although initial studies recognized the importance of different mitochondrial subpopulations, there is a striking lack of direct comparison of intrafibrillar (IF) vs. perinuclear (PN) mitochondria during the development of HF. Here, we use multiple approaches to examine the morphology and functional properties of IF vs. PN mitochondria in pressure overload-induced cardiac remodeling in mice, and in non-failing and failing human cardiomyocytes. We could demonstrate that PN mitochondria from failing cardiomyocytes are more susceptible to changes in mitochondrial membrane potential (ΔΨm), ROS generation and impairment in Ca2+ uptake compared to IF mitochondria at baseline and under physiological stress protocol. We also demonstrated, for the first time, that under normal conditions PN mitochondrial Ca2+ uptake shapes nucleoplasmic Ca2+ transients (CaTs) and prevents nucleoplasmic Ca2+ overload. Loss of PN mitochondria Ca2+ buffering capacity translates into increased nucleoplasmic CaTs and may explain disproportionate rise in nucleoplasmic [Ca2+] in failing cardiomyocytes at increased stimulation frequencies. Therefore, a previously unidentified benefit of restoring the mitochondrial Ca2+ uptake may be normalization of nuclear Ca2+ signaling and alleviation of altered excitation-transcription, which could be an important therapeutic approach to prevent adverse cardiac remodeling. </jats:sec