Mitochondrial Function in Aldosterone-Dependent Human Hypertension

Primary aldosteronism (PA), the most common form of secondary hypertension, is characterized by an increase in cytosolic intracellular Ca2+ concentrations [Ca2+]i and excessive autonomous aldosterone productions. Our aim is to investigate transients and oscillations of [Ca2+]i and to assess mitochondrial function in a human model of CD56+ cells isolated from aldosterone-producing adenoma (APA) and normal surrounding adrenocortical (AAC). Moreover, as PA is associated with abdominal aortic calcification (AAC), we will focus on measuring in vitro calcification propensity of PA and essential hypertensive (EH) plasma patients. We assessed [Ca2+]i in CD56+ APA and AAC in basal conditions and after angiotensin II (Ang II) stimulation using Fura-2 dye, live confocal microscopy, and an ad hoc developed custom-made pipeline to analyze [Ca2+]i signals in terms of frequency and peak parameters as amplitude, full width at half maximum (FWHM) and area under the curve (AUC). As the two-pore domain K+ TASK- 2 channels are under expressed in APA, we used quinidine (20 μM), 20 seconds prior to [Ca2+]i imaging acquisition to examine the effect of the pharmacological inhibition of this channel. In addition, we depleted extracellular Ca2+ to identify the source of Ca2+ transients. In addition, we utilized the Biohybrid assay to assess PA and EH patient’s plasma calcification propensity in vitro using 2 cell models-, namely induced vascular smooth muscle cells (iVSMCs) and human vascular smooth muscle cells (hVSMCs). Resting [Ca2+]i levels were higher in AAC (0.80 AU (25% percentile=0.65; 75% percentile=0.90)) compared to APA cells (0.53 AU (25% percentile=0.33; 75% percentile=0.79)) (p=0.0212). We detected spontaneous Ca2+ activity, during resting conditions, in both cell types without significant difference in peak parameters. Upon Ang II simulation, AAC showed elevated FWHM, amplitude, and AUC peak parameters of first Ca2+ transients. Peak parameters of subsequent Ca2+ oscillations were variable between AAC and APA cells. Ca2+ oscillations frequency and degree of oscillating cells were significantly higher in APA cells than in AAC. In AAC, TASK-2 inhibition with quinidine tended to increase spontaneous activity and altered peak’s parameters, but did not influence frequency of oscillation. Extracellular Ca2+ depletion did not diminish Ca2+ transients. After 24 hours in culture, both AAC and APA cells developed rosette-like cell structure resembling the “glomeruli” of the intact zona glomerulosa clusters. Functional statistics and clustering analysis showed synchronous behavior of cells belonging to the same cluster without significant differences between AAC and APA. Moreover, biohybrid assay results demonstrated elevated extracellular calcification deposition after the stimulation of both VSMC models with PA plasma compared to healthy volunteers. Further, using iVSMCs we observed a significant elevation in calcification deposition compared to EH plasma. Quantitative assessment of [Ca2+]i signals unveiled significant differences in [Ca2+]i dynamics between human AAC and APA cells. These results suggest the concurrent modulation of frequency and amplitude of [Ca2+]i subsequent oscillations as a potential driver of increased rate of aldosterone production. Moreover, we identified an in vitro propensity of plasma from PA patients to drive mineralization deposition, providing a non-invasive diagnostic tool to be used for future personalized cardiovascular risk assessment medicine.