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

Aim: 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.Methods and Results: 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 cmH2O 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%).Conclusions: 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

Cardiomyocyte-Specific Deletion of Orai1 Reveals Its Protective Role in Angiotensin-II-Induced Pathological Cardiac Remodeling

Pathological cardiac remodeling correlates with chronic neurohumoral stimulation and abnormal Ca2+ 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 Ca2+ 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 functionin vivo. Cellular hypertrophyinduced by angiotensin-IIin 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 (Orai1CM–KO). Analysis of cardiac contractility by pressure-volume loops under basal conditions and of cardiac histology did not reveal differences between Orai1CM–KO mice and controls. Moreover, deletion of Orai1in cardiomyocytesin adultmice 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

TRPC channels are not required for graded persistent activity in entorhinal cortex neurons

Abstract: Adaptive behavior requires the transient storage of information beyond the physical presence of external stimuli. This short-lasting form of memory involves sustained ("persistent") neuronal firing which may be generated by cell-autonomous biophysical properties of neurons or/and neural circuit dynamics. A number of studies from brain slices reports intrinsically generated persistent firing in cortical excitatory neurons following suprathreshold depolarization by intracellular current injection. In layer V (LV) neurons of the medial entorhinal cortex (mEC) persistent firing depends on the activation of cholinergic muscarinic receptors and is mediated by a calcium-activated nonselective cation current (ICAN ). The molecular identity of this conductance remains, however, unknown. Recently, it has been suggested that the underlying ion channels belong to the canonical transient receptor potential (TRPC) channel family and include heterotetramers of TRPC1/5, TRPC1/4, and/or TRPC1/4/5 channels. While this suggestion was based on pharmacological experiments and on effects of TRP-interacting peptides, an unambiguous proof based on TRPC channel-depleted animals is pending. Here, we used two different lines of TRPC channel knockout mice, either lacking TRPC1-, TRPC4-, and TRPC5-containing channels or lacking all seven members of the TRPC family. We report unchanged persistent activity in mEC LV neurons in these animals, ruling out that muscarinic-dependent persistent activity depends on TRPC channels

Development of an AAV9-RNAi-mediated silencing strategy to abrogate TRPM4 expression in the adult heart

The cation channel transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective cation channel and acts in cardiomyocytes as a negative modulator of the L-type C

TRPC proteins contribute to development of diabetic retinopathy and regulate glyoxalase 1 activity and methylglyoxal accumulation

Objective: Diabetic retinopathy (DR) is induced by an accumulation of reactive metabolites such as ROS, RNS, and RCS species, which were reported to modulate the activity of cation channels of the TRPC family. In this study, we use Trpc1/4/5/6−/− compound knockout mice to analyze the contribution of these TRPC proteins to diabetic retinopathy. Methods: We used Nanostring- and qPCR-based analysis to determine mRNA levels of TRPC channels in control and diabetic retinae and retinal cell types. Chronic hyperglycemia was induced by Streptozotocin (STZ) treatment. To assess the development of diabetic retinopathy, vasoregression, pericyte loss, and thickness of individual retinal layers were analyzed. Plasma and cellular methylglyoxal (MG) levels, as well as Glyoxalase 1 (GLO1) enzyme activity and protein expression, were measured in WT and Trpc1/4/5/6−/− cells or tissues. MG-evoked toxicity in cells of both genotypes was compared by MTT assay. Results: We find that Trpc1/4/5/6−/− mice are protected from hyperglycemia-evoked vasoregression determined by the formation of acellular capillaries and pericyte drop-out. In addition, Trpc1/4/5/6−/− mice are resistant to the STZ-induced reduction in retinal layer thickness. The RCS metabolite methylglyoxal, which represents a key mediator for the development of diabetic retinopathy, was significantly reduced in plasma and red blood cells (RBCs) of STZ-treated Trpc1/4/5/6−/− mice compared to controls. GLO1 is the major MG detoxifying enzyme, and its activity and protein expression were significantly elevated in Trpc1/4/5/6-deficient cells, which led to significantly increased resistance to MG toxicity. GLO1 activity was also increased in retinal extracts from Trpc1/4/5/6−/− mice. The TRPCs investigated here are expressed at different levels in endothelial and glial cells of the retina. Conclusion: The protective phenotype in diabetic retinopathy observed in Trpc1/4/5/6−/− mice is suggestive of a predominant action of TRPCs in Müller cells and microglia because of their central position in the retention of a proper homoeostasis of the neurovascular unit. Keywords: Diabetic retinopathy, Reactive metabolites, TRPC cation channels, MethylGlyoxal, Vasoregression, Glyoxalase

Novel Nongenetic Murine Model of Hyperglycemia and Hyperlipidemia-Associated Aggravated Atherosclerosis

Objective: Atherosclerosis, the main pathology underlying cardiovascular diseases is accelerated in diabetic patients. Genetic mouse models require breeding efforts which are time-consuming and costly. Our aim was to establish a new nongenetic model of inducible metabolic risk factors that mimics hyperlipidemia, hyperglycemia, or both and allows the detection of phenotypic differences dependent on the metabolic stressor(s). Methods and Results: Wild-typemice were injected with gain-of-function PCSK9D377Y (proprotein convertase subtilisin/kexin type 9) mutant adeno-associated viral particles (AAV) and streptozotocin and fed either a high-fat diet (HFD) for 12 or 20 weeks or a high-cholesterol/high-fat diet (Paigen diet, PD) for 8 weeks. To evaluate atherosclerosis, two different vascular sites (aortic sinus and the truncus of the brachiocephalic artery) were examined in the mice. Combined hyperlipidemic and hyperglycemic (HGHCi) mice fed a HFD or PD displayed characteristic features of aggravated atherosclerosis when compared to hyperlipidemia (HCi HFD or PD) mice alone. Atherosclerotic plaques of HGHCi HFD animals were larger, showed a less stable phenotype (measured by the increased necrotic core area, reduced fibrous cap thickness, and less a-SMA-positive area) and had more inflammation (increased plasma IL-1b level, aortic pro-inflammatory gene expression, and MOMA-2-positive cells in the BCA) after 20 weeks of HFD. Differences between the HGHCi and HCi HFD models were confirmed using RNA-seq analysis of aortic tissue, revealing that significantly more genes were dysregulated in mice with combined hyperlipidemia and hyperglycemia than in the hyperlipidemia-only group. The HGHCi-associated genes were related to pathways regulating inflammation (increased Cd68, iNos, and Tnfa expression) and extracellular matrix degradation (Adamts4 and Mmp14). When comparing HFD with PD, the PD aggravated atherosclerosis to a greater extent in mice and showed plaque formation after 8 weeks. Hyperlipidemic and hyperglycemicmice fed a PD (HGHCi PD) showed less collagen (Sirius red) and increased inflammation (CD68-positive cells) within aortic plaques than hyperlipidemic mice (HCi PD). HGHCi-PD mice represent a directly inducible hyperglycemic atherosclerosis model compared with HFD-fed mice, in which atherosclerosis is severe by 8 weeks. Conclusion: We established a nongenetically inducible mouse model allowing comparative analyses of atherosclerosis in HCi and HGHCi conditions and its modification by diet, allowing analyses of multiple metabolic hits in mice