cardiac-response-to-adrenergic-stimulation-determined-pressure-volume

Cardiac Response to &#946;-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of ...

Ruprecht-Karls-Universität Heidelberg

DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim

TRPM4 proteins form Ca(2+)-activated non selective cation (CAN) channels that affect transmembrane Ca(2+)-influx by determining the membrane potential. Tight control of the intracellular Ca(2+) concentration is essential for mast cell responses. In this study, we analyzed the expression of TRPM4 in peritoneal mast cells (PCMC) as a model for connective tissue type mast cells with respect to FcεRI-evoked calcium changes and the subcellular localization of fluorescently labeled TRPM4 using two viral transduction systems before and following antigen stimulation. Our results show that TRPM4 is expressed in PCMCs, is an essential constituent of the endogenous CAN channels in PCMCs and regulates antigen-evoked increases in intracellular calcium that are significantly enhanced in TRPM4-deficient PCMCs. Compared to PCMCs analyzed before antigen stimulation, the cells depict a substantially increased localization of TRPM4 proteins towards the plasma membrane after FcεRI stimulation. Thus, TRPM4 functions as a limiting factor for antigen evoked calcium rise in connective tissue type mast cells and concurrent translocation of TRPM4 into the plasma membrane is part of this mechanism.

TRPM4-mediated control of FcεRI-evoked Ca(2+) elevation comprises enhanced plasmalemmal trafficking of TRPM4 channels in connective tissue type mast cells.

All three members of the Orai family of cation channels-Orai1, Orai2 and Orai3-are integral membrane proteins that can form store-operated Ca channels resembling endogenous calcium release-activated channels (CRAC) in many aspects. Loss of function studies in human and murine models revealed many functions of Orai1 proteins not only for Ca homeostasis, but also for cellular and systemic functions in many cell types. By contrast, the knowledge regarding the contribution of Orai2 and Orai3 proteins in these processes is sparse. In this study, we report the generation of mouse models with targeted inactivation of the Orai2 gene to study Orai2 function in peritoneal mast cells (PMC), a classical cell model for CRAC channels and Ca-dependent exocytosis of inflammatory mediators. We show that the Ca rise triggered by agonists acting on high-affinity Fc receptors for IgE or on MAS-related G protein-coupled receptors is significantly increased in Orai2-deficient mast cells. Ca entry triggered by depletion of intracellular stores (SOCE) is also increased in Orai2 PMCs at high (2mM) extracellular Ca concentration, whereas SOCE is largely reduced upon re-addtion of lower (0.1mM) Ca concentration. Likewise, the density of CRAC currents, Ca-dependent mast cell degranulation, and mast cell-mediated anaphylaxis are intensified in Orai2-deficient mice. These results show that the presence of Orai2 proteins limits receptor-evoked Ca transients, store-operated Ca entry (SOCE) as well as degranulation of murine peritoneal mast cells but also raise the idea that Orai2 proteins contribute to Ca entry in connective tissue type mast cells in discrete operation modes depending on the availability of calcium ions in the extracellular space.

Deletion of Orai2 augments endogenous CRAC currents and degranulation in mast cells leading to enhanced anaphylaxis.

 Cardiac pressure-volume (PV loop) analysis under β-adrenergic stimulation is a powerful method to simultaneously determine intrinsic cardiac function and β-adrenergic reserve in mouse models. Despite its wide use, several key approaches of this method, which can affect murine cardiac function tremendously, have not been experimentally investigated until now. In this study, we investigate the impact of three lines of action during the complex procedure of PV loop analysis: (i) the ventilation with positive end-expiratory pressure, (ii) the time point of injecting hypertonic saline to estimate parallel-conductance, and (iii) the implications of end-systolic pressure-spikes that may arise under β-adrenergic stimulation.  We performed pressure-volume analysis during β-adrenergic stimulation in an open-chest protocol under Isoflurane/Buprenorphine anesthesia. Our analysis showed that (i) ventilation with 2 cmHO positive end-expiratory pressure prevented exacerbation of peak inspiratory pressures subsequently protecting mice from macroscopic pulmonary bleedings. (ii) Estimations of parallel-conductance by injecting hypertonic saline prior to pressure-volume recordings induced dilated chamber dimensions as depicted by elevation of end-systolic volume (+113%), end-diastolic volume (+40%), and end-diastolic pressure (+46%). Further, using this experimental approach, the preload-independent contractility (PRSW) was significantly impaired under basal conditions (-17%) and under catecholaminergic stimulation (-14% at 8.25 ng/min Isoprenaline), the β-adrenergic reserve was alleviated, and the incidence of ectopic beats was increased >5-fold. (iii) End-systolic pressure-spikes were observed in 26% of pressure-volume recordings under stimulation with 2.475 and 8.25 ng/min Isoprenaline, which affected the analysis of maximum pressure (+11.5%), end-diastolic volume (-8%), stroke volume (-10%), and cardiac output (-11%).  Our results (i) demonstrate the advantages of positive end-expiratory pressure ventilation in open-chest instrumented mice, (ii) underline the perils of injecting hypertonic saline prior to pressure-volume recordings to calibrate for parallel-conductance and (iii) emphasize the necessity to be aware of the consequences of end-systolic pressure-spikes during β-adrenergic stimulation.

Assessment of PEEP-Ventilation and the Time Point of Parallel-Conductance Determination for Pressure-Volume Analysis Under β-Adrenergic Stimulation in Mice.

Pathological cardiac remodeling correlates with chronic neurohumoral stimulation and abnormal Ca signaling in cardiomyocytes. Store-operated calcium entry (SOCE) has been described in adult and neonatal murine cardiomyocytes, and Orai1 proteins act as crucial ion-conducting constituents of this calcium entry pathway that can be engaged not only by passive Ca store depletion but also by neurohumoral stimuli such as angiotensin-II. In this study, we, therefore, analyzed the consequences of Orai1 deletion for cardiomyocyte hypertrophy in neonatal and adult cardiomyocytes as well as for other features of pathological cardiac remodeling including cardiac contractile function in vivo. Cellular hypertrophy induced by angiotensin-II in embryonic cardiomyocytes from Orai1-deficient mice was blunted in comparison to cells from litter-matched control mice. Due to lethality of mice with ubiquitous Orai1 deficiency and to selectively analyze the role of Orai1 in adult cardiomyocytes, we generated a cardiomyocyte-specific and temporally inducible Orai1 knockout mouse line (Orai1). Analysis of cardiac contractility by pressure-volume loops under basal conditions and of cardiac histology did not reveal differences between Orai1 mice and controls. Moreover, deletion of Orai1 in cardiomyocytes in adult mice did not protect them from angiotensin-II-induced cardiac remodeling, but cardiomyocyte cross-sectional area and cardiac fibrosis were enhanced. These alterations in the absence of Orai1 go along with blunted angiotensin-II-induced upregulation of the expression of Myoz2 and a lack of rise in angiotensin-II-induced STIM1 and Orai3 expression. In contrast to embryonic cardiomyocytes, where Orai1 contributes to the development of cellular hypertrophy, the results obtained from deletion of Orai1 in the adult myocardium reveal a protective function of Orai1 against the development of angiotensin-II-induced cardiac remodeling, possibly involving signaling via Orai3/STIM1-calcineurin-NFAT related pathways.

Rebekka Medert

Pharmakologisches Institut

Ruprecht-Karls-Universität Heidelberg

Cardiac Response to β-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis