A cell atlas of thoracic aortic perivascular adipose tissue: a focus on mechanotransducers.

Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. However, major gaps remain in our understanding of the cells present in PVAT, as well as how different cells contribute to mechanotransduction. We hypothesized that snRNA-seq would reveal the expression of mechanotransducers, and test one (PIEZO1) to illustrate the expression and functional agreement between single-nuclei RNA sequencing (snRNA-seq) and physiological measurements. To contrast two brown tissues, subscapular brown adipose tissue (BAT) was also examined. We used snRNA-seq of the thoracic aorta PVAT (taPVAT) and BAT from male Dahl salt-sensitive (Dahl SS) rats to investigate cell-specific expression mechanotransducers. Localization and function of the mechanostransducer PIEZO1 were further examined using immunohistochemistry (IHC) and RNAscope, as well as pharmacological antagonism. Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNA-seq, identifying eight major cell types expected and one unexpected (nuclei with oligodendrocyte marker genes). Cell-specific differential gene expression analysis between taPVAT and BAT identified up to 511 genes (adipocytes) with many (≥20%) being unique to individual cell types. Piezo1 was the most highly, widely expressed mechanotransducer. The presence of PIEZO1 in the PVAT but not the adventitia was confirmed by RNAscope and IHC in male and female rats. Importantly, antagonism of PIEZO1 by GsMTX4 impaired the PVAT's ability to hold tension. Collectively, the cell compositions of taPVAT and BAT are highly similar, and PIEZO1 is likely a mechanotransducer in taPVAT.NEW & NOTEWORTHY This study describes the atlas of cells in the thoracic aorta perivascular adipose tissue (taPVAT) of the Dahl-SS rat, an important hypertension model. We show that mechanotransducers are widely expressed in these cells. Moreover, PIEZO1 expression is shown to be restricted to the taPVAT and is functionally implicated in stress relaxation. These data will serve as the foundation for future studies investigating the role of taPVAT in this model of hypertensive disease.

Deep transcriptomic profiling of Dahl salt-sensitive rat kidneys with mutant form of Resp18.

Expression of Regulated endocrine specific protein 18 (Resp18) is localized in numerous tissues and cell types; however, its exact cellular function is unknown. We previously showed that targeted disruption of the Resp18 locus in the Dahl SS (SS) rat (Resp18mutant) results in higher blood pressure (BP), increased renal fibrosis, increased urinary protein excretion, and decreased mean survival time following a chronic (6 weeks) 2% high salt (HS) diet compared with the SS rat. Based on this prominent renal injury phenotype, we hypothesized that targeted disruption of Resp18 in the SS rat promotes an early onset hypertensive-signaling event through altered signatures of the renal transcriptome in response to HS. To test this hypothesis, both SS and Resp18mutant rats were exposed to a 7-day 2% HS diet and BP was recorded by radiotelemetry. After a 7-day exposure to the HS diet, systolic BP was significantly increased in the Resp18mutant rat compared with the SS rat throughout the circadian cycle. Therefore, we sought to investigate the renal transcriptomic response to HS in the Resp18mutant rat. Using RNA sequencing, Resp18mutant rats showed a differential expression of 25 renal genes, including upregulation of Ren. Upregulation of renal Ren and other differentially expressed genes were confirmed via qRT-PCR. Moreover, circulating renin activity was significantly higher in the Resp18mutant rat compared with the WT SS rat after 7 days on HS. Collectively, these observations demonstrate that disruption of the Resp18 gene in the SS rat is associated with an altered renal transcriptomics signature as an early response to salt load.

CACNB2 is associated with aberrant RAS-MAPK signaling in hypertensive Dahl Salt-Sensitive rats.

Several independent genome-wide association studies (GWAS) have indicated that calcium (Ca2+) voltage-gated channel auxiliary subunit beta 2 (CACNB2) an L-type Ca2+ channel (LTCC) associated protein has strong association with hypertension. However, the molecular mechanism of CACNB2 and its role in the pathophysiology of hypertension is not clear. To address this knowledge gap, we utilized in vitro and in vivo approaches using HEK293 cells and genetically hypertensive, Dahl Salt-Sensitive (SS) rats. We demonstrated that CACNB2 over-expression in HEK293 cells triggers cell proliferation via an up-regulation of the RAS-MAPK pathway compared to non-transfected cells. These effects were likely independent of LTCC activity as treatment with nifedipine, a well-known LTCC blocker, in CACNB2 overexpressing cells failed to inhibit the RAS-MAPK pathway gene expressions or show an effect on apoptosis marker gene expression. Furthermore, the expression level of CACNB2 was up-regulated in the high salt (HS) diet fed SS rat kidneys compared to low salt diet (LS) fed group. Similar to our in vitro observation the RAS-MAPK mRNA levels were increased in HS fed SS rat kidneys, compared to LS fed group. Collectively, our data suggest that CACNB2 is associated with the increase in RAS-MAPK gene expressions and lead us to speculate that in addition to its role in regulating LTCC α1-subunit trafficking, CACNB2 might lead to aberrant RAS activation, which is one of the key cascade associated with hypertension.

A Cell Atlas of Thoracic Aortic Perivascular Adipose Tissue: a focus on mechanotransducers.

Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. To examine the cell-specificity of recognized mechanotransducers we used single nuclei RNA sequencing (snRNAseq) of the thoracic aorta PVAT (taPVAT) from male Dahl SS rats compared to subscapular brown adipose tissue (BAT). Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNAseq, identifying 8 major cell types expected and one unexpected (nuclei with oligodendrocyte marker genes). Cell-specific differential gene expression analysis between taPVAT and BAT identified up to 511 genes (adipocytes) with many (≥20%) being unique to individual cell types. Piezo1 was the most highly, widely expressed mechanotransducer. Presence of PIEZO1 in the PVAT was confirmed by RNAscope® and IHC; antagonism of PIEZO1 impaired the PVAT's ability to hold tension. Collectively, the cell compositions of taPVAT and BAT are highly similar, and PIEZO1 is likely a mechanotransducer in taPVAT.

Effects of zamicastat treatment in a genetic model of salt-sensitive hypertension and heart failure.

Hyperactivity of sympathetic nervous system plays an important role in the development and progression of cardiovascular diseases. An approach to mitigate the enhanced sympathetic nervous system drive is restricting the biosynthesis of noradrenaline via inhibition of the enzyme dopamine ß-hydroxylase (DßH), that catalyzes the hydroxylation of dopamine to noradrenaline in sympathetic nerves. The aim of the present study was to evaluate the effects of zamicastat, a novel DßH inhibitor that decreases noradrenaline and increases dopamine levels in peripheral sympathetically innervated tissues, on the hemodynamic and cardiometabolic parameters in salt-induced hypertension and heart failure in the Dahl salt-sensitive (SS) rat. Zamicastat (10, 30 and 100 mg/kg body weight) was tested acutely against salt-induced hypertension in the Dahl SS rat. Chronic zamicastat treatment (30 mg/kg/day) was evaluated against salt-induced cardiac hypertrophy and biomarkers of cardiometabolic risk and inflammation in Dahl SS rats and upon the survival rate in aged Dahl SS rats fed a high-salt diet. The reduction in the sympathetic tone attained with zamicastat shaped a dose- and time-dependent effect on blood pressure. Prolonged treatment with zamicastat ameliorated end-organ damage, metabolic syndrome and inflammation hallmarks in hypertensive Dahl SS rats. Survival rate of Dahl SS rats fed a high-salt diet demonstrated that zamicastat increased median survival of Dahl SS rats fed a high-salt diet. The use of DßH inhibitors, like zamicastat, is a promising approach to treat hypertension, heart failure and cardiovascular diseases where a reduction in the sympathetic tone has beneficial effects.

Progression of glomerular filtration rate reduction determined in conscious Dahl salt-sensitive hypertensive rats.

Sequential changes in glomerular filtration rate during development of hypertension in the conscious Dahl salt-sensitive rats were determined using a new method for measurement. Using a miniaturized device, disappearance curves of fluorescein isothiocyanate-sinistrin were measured by transcutaneous excitation and real-time detection of the emitted light through the skin. Rats with implanted femoral venous catheters (dye injection and sampling) and carotid catheters (mean arterial pressure by telemetry) were studied, while maintained on a 0.4% NaCl diet and on days 2, 5, 7, 14, and 21 after switching to 4.0% (high-salt [HS]) diet. A separate group of rats were maintained on 0.4% for 21 days as a time control. Mean arterial pressure rose progressively from the last day of 0.4% (130±2 mm Hg) reaching significance by day 5 of HS and averaged 162±7 mm Hg by day 21. Urine albumin excretion was significantly elevated (×3) by day 7 of HS in Dahl salt-sensitive rats. Glomerular filtration rate reduced on day 14 of HS falling from 1.53±0.06 mL/min per 100 g body weight to 1.27±0.04. By day 21, glomerular filtration rate had fallen 28% to 1.1±0.04 mL/min per 100 g (t(1/2) 28.4±1.1 minute.) No significant reductions of creatinine clearance were observed throughout the study in response to HS demonstrating the insensitivity of creatinine clearance measurements even with creatinine measured using mass spectrometry. We conclude that the observed reduction of glomerular filtration rate was a consequence and not a cause of the hypertension and that this noninvasive approach could be used in these conscious Dahl salt-sensitive rats for a longitudinal assessment of renal function.